Methods of determining a treatment protocol for and/or a prognosis of a patient&#39;s recovery from a brain injury

ABSTRACT

The present invention, in some embodiments, generally relates to methods of determining a treatment protocol for and/or a prognosis of a patient&#39;s recovery from a brain injury. In some embodiments, the brain injury results from a hypoxic event. In some embodiments, methods are provided for determining a measure of the concentration of tau protein in a patient sample containing or suspected of containing tau protein.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/269,142, filed Sep. 19, 2016, and entitled “METHODS OF DETERMINING ATREATMENT PROTOCOL FOR AND/OR A PROGNOSIS OF A PATIENT'S RECOVERY FROM ABRAIN INJURY,” which is a continuation of U.S. patent application Ser.No. 14/111,326, filed Jun. 24, 2014, and entitled “METHODS OFDETERMINING A TREATMENT PROTOCOL FOR AND/OR A PROGNOSIS OF A PATIENT′SRECOVERY FROM A BRAIN INJURY,” which is a national stage ofInternational Patent Application Ser. No. PCT/US2012/033343, filed Apr.12, 2012, and entitled “METHODS OF DETERMINING A TREATMENT PROTOCOL FORAND/OR A PROGNOSIS OF A PATIENT′S RECOVERY FROM A BRAIN INJURY,” whichclaims priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication Ser. No. 61/474,315, filed Apr. 12, 2011, and entitled“METHODS OF DETERMINING A TREATMENT PROTOCOL FOR AND/OR A PROGNOSIS OF APATIENT'S RECOVERY FROM A BRAIN INJURY RESULTING FROM A HYPDXIC EVENT,”and claims priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication Ser. No. 61/524,693, filed Aug. 17, 2011, and entitled“METHODS OF DETERMINING A TREATMENT PROTOCOL FOR AND/OR A PROGNOSIS OF APATIENT'S RECOVERY FROM A BRAIN INJURY,” each of which are incorporatedherein by reference in their entireties for all purposes.

FIELD OF THE INVENTION

The present invention generally relates, in some embodiments, to methodsof determining a treatment protocol for and/or a prognosis of apatient's recovery from a brain injury. In some embodiments, the braininjury results from a hypoxic event. In some embodiments, methods areprovided for determining a measure of the concentration of tau proteinin a patient sample containing or suspected of containing tau protein.

BACKGROUND OF THE INVENTION

A brain injury in a human may be caused by any number of events orconditions. In some cases, a brain injury may be caused by externalmechanical force, such as rapid acceleration or deceleration, impact,blast waves, or penetration by a projectile. This type of acquired braininjury is generally known as traumatic brain injury. Another type ofacquired brain injury involves biochemical forces, such as oxygendeprivation (hypoxia). Hypoxia generally refers to a deficiency in theamount of oxygen reaching body tissues or a condition of insufficientlevels of oxygen in tissue or blood. Oxygen deprivation to the brainresults in neuronal damage and death, which is in turn related to theextent of long term brain dysfunction. The concentration of certainbiomarkers may become elevated as a result of neuronal damage and death.For example, tau proteins are associated with microtubules and localizedin the axonal compartment of neurons. Tau is known to be elevated in thecerebrospinal fluid (CSF) of patients with neurodegenerative disease andhead injuries. However, since such biomarkers must diffuse across theblood brain barrier, they may be present in the blood in proportion inextremely low concentrations that are not reliably measurable by typicalconventional immunoassays. While the concentration of some biomarkers inthe brain and central nervous system are known to increase with hypoxicevents, the increased concentration has not been correlated withspecific diagnostic indications and/or methods of treatment. Inaddition, while some methods exist for determining a brain injury in apatient and/or determining a course of treatment following a braininjury, many of the known methods are costly (e.g., magnetic resonanceimaging) and/or provide unclear results and/or predictors. Accordingly,improved methods are needed.

SUMMARY OF THE INVENTION

In some embodiments, a method for determining a measure of theconcentration of tau protein in a patient sample containing or suspectedof containing tau protein is provided comprising performing an assay todetermine a measure of the concentration of tau protein in the sample,wherein the limit of detection of tau protein of the assay is less thanabout 0.2 pg/mL.

In some embodiments, a method of determining a treatment protocol forand/or a prognosis of a patient's recovery from a brain injury isprovided comprising performing an assay on a blood sample from thepatient and/or plasma and/or serum derived from the blood sample todetermine a measure of the concentration of tau protein in the sample;and determining a prognosis of the patient's recovery from the braininjury and/or a method of treatment based at least in part on themeasured concentration of tau protein present in the sample.

In some embodiments, a method of determining a treatment protocol forand/or a prognosis of a patient's recovery from a brain injury isprovided comprising determining a prognosis of the patient's recoveryfrom the brain injury and/or a method of treatment based at least inpart on a measured concentration of tau protein present in a patientsample, wherein the measured concentration has been determined byperforming an assay on the patient sample, which comprises a bloodsample from the patient and/or plasma and/or serum derived from theblood sample, to determine the measure of the concentration of tauprotein in the sample.

In some embodiments, a method for performing an assay and providing datafor determining a treatment protocol for and/or a prognosis of apatient's recovery from a brain injury is provided comprising performingan assay on a blood sample from the patient and/or plasma and/or serumderived from the blood sample to determine a measure of theconcentration of tau protein in the sample; and providing data from theassay to enable determining a prognosis of the patient's recovery fromthe brain injury and/or a method of treatment based at least in part onthe measured concentration of tau protein present in the sample.

In some embodiments, a method of determining a treatment protocol forand/or a prognosis of a patient's recovery from a brain injury isprovided comprising determining a measure of the concentration of tauprotein in each of a plurality of samples obtained from the patientfollowing the brain injury; and determining a prognostic of thepatient's recovery from the brain injury and/or a method of treatmentbased at least in part on the measured concentration tau protein presentin the sample.

In some embodiments, a method of determining a method of treatment forand/or a prognosis of a patient's recovery from a brain injury isprovided comprising (a) performing an assay on each of a plurality ofsamples obtained from the patient following the brain injury todetermine the measured concentration of tau protein in each of thesamples, wherein the plurality of samples are obtained from the patientover a period of time of at least about 48 hours; (b) determining thearea under the curve of a graph of the tau protein concentration in theplurality of samples versus time, wherein the area is determined for theentire time period and/or for a second peak in the tau proteinconcentration; and (c) determining a prognosis of the patient's recoveryfrom the brain injury and/or a method of treatment based at least inpart on the area under the curve for the entire time period and/or thesecond peak in the tau protein concentration determined in step (b).

In some embodiments, a method of determining a method of treatment forand/or a prognosis of a patient's recovery from a brain injury isprovided comprising determining a prognosis of the patient's recoveryfrom the brain injury and/or a method of treatment based at least inpart on the area under the curve of a graph of the tau proteinconcentration in the plurality of samples versus time, wherein the areais determined for the entire time period and/or for a second peak in thetau protein concentration, which has been determined by (a) performingan assay on each of a plurality of samples obtained from the patientfollowing the brain injury to determine the measured concentration oftau protein in each of the samples, wherein the plurality of sampleshave been obtained from the patient over a period of time of at leastabout 48 hours; and (b) determining the area under the curve of a graphof the tau protein concentration in the plurality of samples versus timefor the entire time period and/or for a second peak in the tau protein.

In some embodiments, a method for performing an assay and providing datafor determining a method of treatment for and/or a prognosis of apatient's recovery from a brain injury is provided comprising (a)performing an assay on each of a plurality of samples obtained from thepatient following the brain injury to determine the measuredconcentration of tau protein in each of the samples, wherein theplurality of samples are obtained from the patient over a period of timeof at least about 48 hours; (b) determining the area under the curve ofa graph of the tau protein concentration in the plurality of samplesversus time, wherein the area under the curve is determined for theentire time period and/or for a second peak in the tau proteinconcentration; and (c) providing data derived in steps (a) and (b) toenable determining a prognosis of the patient's recovery from the braininjury and/or a method of treatment based at least in part on the areaunder the curve determined for the entire time period and/or for asecond peak in the tau protein concentration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a schematic flow diagram depicting one embodiment of steps(A-D) for performing an exemplary method of the present invention;

FIG. 1b is a schematic flow diagram depicting one embodiment of steps(A-D) for performing an exemplary method of the present invention;

FIG. 2 show plots of serum tau concentrations for numerous patientsfollowing resuscitation from cardiac arrest having a-c) a poor outcomeand d) a good outcome;

FIG. 3 shows plots of receiver operating characteristics (ROC) curvesand areas under the curve values of tau protein concentration versustime for a) the first 24 hours, b) all serial samplings, and c) thesecondary tau peak only;

FIG. 4 illustrates six naturally occurring isoforms of tau proteins.

Other aspects, embodiments, and features of the invention will becomeapparent from the following detailed description when considered inconjunction with the accompanying drawings. The accompanying figures areschematic and are not intended to be drawn to scale. For purposes ofclarity, not every component is labeled in every figure, nor is everycomponent of each embodiment of the invention shown where illustrationis not necessary to allow those of ordinary skill in the art tounderstand the invention. All patent applications and patentsincorporated herein by reference are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control.

DETAILED DESCRIPTION

The present invention generally relates to methods of determining atreatment protocol for and/or a prognosis of a patient's recovery from abrain injury. In some embodiments, the brain injury may result from ahypoxic event. In some embodiments, methods are provided for determininga measure of the concentration of tau protein in a patient samplecontaining or suspected of containing tau protein. The subject matter ofthe present invention involves, in some cases, interrelated products,alternative solutions to a particular problem, and/or a plurality ofdifferent uses of one or more systems and/or articles.

In some embodiments, a method of the present invention comprisesdetermining a measure of the concentration of at least one biomarker inone or more samples obtained from a patient following a brain injury. Insome embodiments, a method of the present invention comprisesdetermining a measure of the concentration of at least one biomarker inone or more samples obtained from a patient following the brain injury(e.g., optionally resulting from a hypoxic event). A prognosticindication of the patient's recovery and/or determining a course oftreatment (e.g., from the brain injury, optionally resulting from ahypoxic event) may be based at least in part on the measure of theconcentration of the at least one biomarker present in the one or moresamples. It should be understood, that while much of the discussionbelow is directed to methods involving the analysis of more than onesample, this is by way of example only, and similar methods may beemployed wherein only a single sample is employed.

As will be known to those of ordinary skill in the art, the term“hypoxia” generally refers to a deficiency in the amount of oxygenreaching body tissues or a condition of insufficient levels of oxygen intissue or blood. Hypoxia at a cellular level develops when delivery ofoxygen to cell mitochondria slows as the partial pressure gradient fromcapillaries to tissues decreases. As the delivery of oxygen decreases,aerobic metabolism stops and less efficient anaerobic pathways ofglycolysis become responsible for the production of cellular energy. Theend result is an increase in cellular concentrations of sodium, calcium,and hydrogen ions which may lead to cell death.

Oxygen deprivation to the brain results in neuronal damage and death.The extent of neuronal damage and death in turn relates to the extent oflong term brain dysfunction, as can be assessed using standard criteria(such as Cerebral Performance Category, CPC rating, or like criteria).Severe hypoxia can result in a patient's death and/or an irreversiblebrain injury (e.g., resulting in the patient being in a vegetativestate). Hypoxic events may be global (e.g., due to low oxygen content inthe blood) or focused (e.g., affecting only an area of the brain).Causes of hypoxia include, but are not limited to, local asphyxia (e.g.,caused by smoke inhalation), carbon monoxide poisoning and/or toxicity,cardiac arrest, choking, drowning, high altitudes, strangulation, anischemic event, thrombosis, arterial embolism, hemorrhage, swelling ofthe brain, stroke, physical trauma and/or physical injury (e.g., blunttrauma to the head), arteriosclerosis, and/or atherosclerosis. In somecases, the event may be myocardial infarction, myocardial ischemia,and/or transient ischemic attack.

Hypoxic conditions can lead to the production and/or change in theconcentration of certain biomarkers. That is, the concentration ofcertain biomarkers increase or decrease following the hypoxic event. Forexample, the production of the proteolytic products of β-amyloidprecursor protein has been found to become elevated in the brain andcentral nervous system under hypoxic condition. The increasedconcentration is theorized to be due to a hypoxia-inducible factor(HIF-1) that promotes the production of beta-amyloid peptides fromamyloid precursor protein, a membrane protein concentrated in neuronalsynapses. A cascade of biomarkers, such as tau proteins, is generated inthe brain in proportion to the extent of hypoxia. Such biomarkers couldin turn diffuse across the blood brain barrier and into the blood inproportion to the extent of hypoxia, and may be generally found in lowabundance. The ability to determine a change in the concentration of abiomarker in a plurality of samples (or a single sample) obtained from apatient following a hypoxic event can, in some embodiments, becorrelated with a prognostic indication of the patient's recovery from abrain injury and/or used to determine a method of treatment. In somecases, sample(s) of the patient's cerebrospinal fluid (CSF) may beobtained and analyzed to determine the concentration and/or a change inthe concentration of the biomarker. In some cases, however, it isadvantageous to determine the level of a biomarker in the blood of apatient as compared to CSF, as blood sampling is generally less invasiveand may result in fewer complications as compared to CSF sampling.However, many of the biomarkers that are present in the CSF have a slowrate of transmission across and/or a high barrier of transportationacross the blood-brain barrier (BBB) and thus, the concentration of thebiomarker in the patient's blood may be sufficient lowered as comparedto the concentration in CSF to make it difficult or impossible toaccurately determine using typically employed conventional immunoassays.Accordingly, assay methods which have very low limits of quantification(LOQ) and/or limits of detection (LOD) are generally necessary todetermine a measure of the concentration of a biomarker in the patient'sblood to provide statistically significant and/or meaningful results. Insome embodiments, the methods of the present invention make use ofmethods having very low LODs and/or LOQs (e.g., in the low pg/mL range)to determine a measure of the concentration of a biomarker in asample(s) obtained from a patient following a hypoxic event. Variousparameters related to the changes in the concentration of the biomarkerin the samples (e.g., blood samples) may be correlated with a prognosticindication and/or a method of treatment following a hypoxic event.Correlations (e.g., between the concentration and prognosticindication(s) and/or between the concentration and method(s) oftreatment) have been discovered and/or are now discoverable due torecent advancements in technology which allow for the determination ofthe low concentrations of biomarkers in bodily fluids with sufficientaccuracy and precision, thus allowing for the variations inconcentration to be statistically significant and therefore, diagnostic.

It should be noted, that while many of the embodiments described hereinfocus on brain injuries caused by hypoxic events, this is by no waylimiting, and in some embodiments, the brain injury may be caused byother events, for example, traumatic brain injuries wherein the force issuch that the skull fractures causing mechanical damage to the brain. Insome cases, a traumatic brain injury may be caused by externalmechanical force, such as rapid acceleration or deceleration, impact,blast waves, or penetration by a projectile.

In embodiments where a plurality of samples are obtained from a patient,the samples may be obtained from a patient over any suitable period oftime. Generally, the period of time may be selected such that theconcentration of a biomarker in the samples becomes statisticallysignificant and/or a trend is observable (e.g., an increase and/ordecrease in the concentration). For biomarkers which are analyzed inblood samples, the period of time over which a plurality of samples areobtained from the patient may account for any lag time required for thebiomarker to cross the BBB. Non-limiting examples of suitable periods oftime in which the samples may be obtained from the patient include 1hour, 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours,18 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 4 days, 5days, 6 days, 7 days, or more. In some cases, the duration of time ofsample collection time is at least 60 hours, or at least 72 hours. Insome cases, the duration of time of sample collection is between 12hours and 7 days, or between 24 hours and 4 days, or between 2 days and4 days, or between 3 days and 4 days. The first sample may be obtainedfrom the patient without a short timeframe following the brain injury.For example, the first sample may be obtained from the patient within 1hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 10 hours, or12 hours of the brain injury. In some cases, the first sample isobtained within 6 hours of the brain injury. In some embodiments, afirst sample is obtained from the patient within 6 hours of thesuspected brain injury, and at about 1, about 2, about 6, about 12,about 24, about 48, and about 72 hours, following the first sampling. Insome embodiments, additional samples are obtained at about 96 and/or atabout 108 hours following the first sampling.

Any number of samples (e.g., one or more) may be obtained from thepatient over the time period of sample collection. Generally, theminimum number of samples obtained is such that a trend (e.g., anincrease or decrease) in the concentration of the biomarker isobservable. Non-limiting examples of the number of samples that areobtained from the patient (e.g., during the prescribed collection time)is at least about bout 1, about 2, at least about 3, at least about 4,at least abut 5, at least about 6, at least about 7, at least about 8,at least about 9, at least about 10, at least about 12, at least about15 or more. In some cases, the number of samples obtained from thepatient is between 2 and 20, between 5 and 15, or between 5 and 10.

The sample(s) obtained from the patient may be from any suitable bodilysource. In some cases, the samples are CSF fluid samples. In some cases,the samples are not CSF fluid samples. In some cases, the samples areblood or blood products (e.g., whole blood, plasma, serum, etc.). Inother cases, the samples may be urine or saliva samples. In someembodiments, the samples may be analyzed directly (e.g., without theneed for extraction of the biomarker from the fluid sample) and/or withdilution (e.g., addition of a buffer or agent to the sample). Generally,each of the samples obtained from the patient is collected usingsubstantially similar procedures (e.g., to ensure minimal variationbetween samples based on sample collection methods). Those of ordinaryskill in the art will be aware of suitable systems and methods forobtaining a sample from a patient.

Each of or substantially all of the samples may be analyzed using anassay method (e.g., as described herein) to determine a measure of theconcentration of at least one biomarker in each, a subset of orsubstantially all of the samples. In some cases, the methods comprisedetermining a measure of the concentration of a single biomarker in thesamples. In other cases, a method comprises determining a measure of theconcentration of more than one biomarker in each, a subset of orsubstantially all of the samples. For example, a measure of theconcentration of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, biomarkers may bedetermined in the samples.

In some embodiments, the methods of the present invention comprisedetermining a prognostic indication based at least in part on themeasured concentration of the at least one biomarker in the samples. Insome cases, the prognostic indication may be correlated with standardcriteria employed to define long-term brain dysfunction and/or injury.Those of ordinary skill in the art will be aware of such criteria, forexample, cerebral performance category ratings (“CPC rating”), or morespecifically, Glasgow-Pittsburgh cerebral performance category ratings(or scale) (e.g., see Teasdale G, Jennett B (1974); Assessment of comaand impaired consciousness; Lancet 2 (7872): 81-84). The CPC scaleranges from 1 to 5, with 1 representing a slight possibility ofneurological deficit and 5 representing severe deficit and/or death. Insome methods of the present invention, the prognostic indication of thepatient's recovery from the brain injury is classified as either “good”(e.g., correlating to a CPC score of 1 or 2) corresponding to a highlikelihood of recovery and/or returning to independent living, or “poor”(e.g., correlating to a CPC score of 3, 4, or 5) corresponding to littlepossibility of a full recovery and resulting in assisted living and/ordeath.

In the CPC scale, a rating of 1 is generally classified as good cerebralperformance. The patient is conscious and alert, is able to work, butmay have mild neurological or psychological deficit. A rating of 2 isgenerally classified as having moderate cerebral disability. The patientis conscious and has sufficient cerebral function for independentactivities of daily life, and is generally able to work in shelteredenvironment. A rating of 3 corresponds to severe cerebral disability.While the patient is conscious, they generally depend on others fordaily support because of impaired brain function. The patient may haveabilities ranging from ambulatory state to severe dementia or paralysis.A rating of 4 corresponds to a coma or vegetative state. The patient isgenerally unaware, even if they appear awake (e.g., the patient is in avegetative state) without interaction with environment and is cerebralunresponsive. A rating of 5 refers to brain death, associated withapnea, areflexia, and/or EEG silence. The CPC scale is summarized inTable 1.

TABLE 1 CPC Scale Summary CPC Score Description 1 Conscious and alertwith normal function or only slight disability 2 Conscious and alertwith moderate disability 3 Conscious with severe disability 4 Comatoseor persistent vegetative state 5 Brain dead or death from other causes

Other non-limiting examples of suitable criteria include the “scale g”criteria (e.g., see Dekaban A S, Robinson C E.; Application of a newrating scale of brain dysfunction to monitoring rehabilitation in 65patients with severe head injury, Bull Clin Neurosci., 1984; 49, 82-92),the “Rancho Los Amigos Scale,” and the “Disabilities Rating Scale.”

In addition to the biomarkers specifically mentioned herein, those ofordinary skill will be aware of other suitable biomarkers to use inconnection with the methods described herein. As described herein, thebiomarker generally undergoes a change in concentration as a result of ahypoxic event. For example, the concentration of the biomarker mayincrease or decrease as a result of the brain injury. Non-limitingexamples of biomarkers include neuron specific neuronal enolase (NSE),β-site aPP-cleaving enzyme 1 (BACe1), S100B, myelin basic protein (MBP),growth associated protein 43, glutamine synthetase, glial fibrillaryacid protein (GFAP), glycine transporter (e.g., GLYT1, GLYT2), neuronspecific glycoprotein (e.g., GP50), calpain, neurofibrillary protein,heat shock protein 72, beta-amyloid precursor proteins, calbindin D-28K,proteolipid protein, myeline associated glycoprotein, neurofilament H,creatine kinase protein (e.g., CK-BB), tau proteins (includingphosphorylated taus such as p-tau-81 or p-tau-231), and endotheliummembrane proteins (e.g., thrombomodulin).

In some cases, the biomarker is a tau protein. Various forms and/orcombinations of tau proteins may be contemplated for use as a targetbiomarker with the methods described herein, include isoforms and shortisoforms, for example, ranging from tau 23 (352a.a, “0N3R”, wherein Rindicates the number of repeats and N indicates the number or aminoterminal inserts, as will be understood by those of ordinary skill inthe art) to tau 40 (441a.a, “2N4R”). There are six knownnaturally-occurring tau proteins, the sequences of which are well knownin the art. The six tau proteins include tau 23 (352, 0N3R), tau 24(383, 0N4R), tau 37 (381, 1N3R), tau 34 (412, 1N4R), tau 39 (410, 2N3R),tau 40 (441, 2N4R), and/or combinations thereof (e.g., see FIG. 4). Insome cases, at least some of the tau proteins may be phosphorylated.

Those of ordinary skill in the art will understand that determination ofa biomarker in a sample may comprise determining the concentration asingle isoform of a biomarker, or alternatively, may comprisedetermining the concentration of a plurality of isoforms of thebiomarker. For example, with respect to tau proteins, in some cases, theconcentration of tau protein employed in the algorithms and methodsdescribed herein may be the concentration of a single isoform of tauprotein in the sample, or alternatively, the concentration of tauprotein employed in the algorithms and methods described herein may bethe concentration of a plurality of forms of tau proteins in the sample.

The plurality of samples obtained from the patient may be analyzed(e.g., using an assay method as described herein) to determine a measureof the concentration of the at least one biomarker in each of thesamples (or a single sample). A prognostic indication and/or a method oftreatment may be determined based at least in part on the measure of theconcentration of the biomarker in each of the plurality of samples. Thedata may be analyzed using a variety of techniques, as described herein,and a prognostic indication for recovery from a brain injury (e.g., a“good” outcome or a “poor” outcome), or a specific method of treatmentmay be determined based on the results.

In some embodiments, the measure of the concentration of the at leastone biomarker for each of the plurality of samples obtained from thepatient may be plotted on a graph of concentration versus time (e.g., inhours), and one or more parameters can be obtained from the graph andused to determine the method of treatment and/or the prognosticindication. Non-limiting examples of parameters that may be determinedand/or employed include baseline biomarker concentration, increase inbiomarker concentration, duration of rise of biomarker concentration,maximum slope of increasing biomarker concentration, rate of change ofbiomarker concentration, area under the curve and/or magnitude of thefold increase of biomarker concentration. Each of the parameters andtheir determination will now be described in detail, followed by adescription of possible data analysis and methods useful or potentiallyuseful to determine suitable correlations between the measure of theconcentration determined in the samples and a treatment/prognosticindication. The measure of the concentration of the biomarker may bedetermined/displayed in any suitable unit. In some cases, the measure ofthe concentration is determined/displayed in pg/mL.

The term “baseline biomarker concentration” refers to the concentrationof the biomarker generally present in a fluid sample from a normalpatient (e.g., prior to or unexposed to a hypoxic event). The baselinebiomarker concentration can be determined using a variety of methodswhich will be commonly known and understood by those of ordinary skillin the art. In some cases, the baseline biomarker concentration can bedetermined by averaging the value of a biomarker present in a populationof control patients (e.g., patients who have not experienced a hypoxicevent). This may be useful in embodiments where the baselineconcentration of the biomarker is substantially the same for a givenpopulation of individuals (e.g., based on age, gender, medicalcondition, etc.). However, many of the methods described herein requireaccurate determination of extremely low concentrations of the at leastone biomarker in samples obtained from a patient, and accordingly, ageneral baseline concentration of a biomarker (e.g., based on a samplingof a population) may not provide enough accuracy and/or precision togive useful results. Accordingly, a measure of the baseline biomarkerconcentration, in some embodiments, may be determined for eachindividual patient. In some embodiments, the baseline biomarkerconcentration may be set to zero, e.g., in embodiments where theconcentration of the biomarker is zero or essentially zero in normalpatients.

In some cases, the baseline biomarker concentration for each individualis equal to the measure of the concentration of the biomarker in thefirst sample obtained from the patient following the hypoxic event. Inother cases, if available, a baseline biomarker concentration is themeasure of the concentration of the biomarker present in a sampleobtained from the patient prior to the hypoxic event. In yet othercases, the baseline biomarker concentration is the average concentrationof the biomarker determined in a plurality of samples taken from thepatient immediately or substantially immediately following the hypoxicevent (e.g., prior to the concentration of the biomarker increasing dueto the hypoxic event). That is, the concentration of the biomarker inthe plurality of samples obtained from the patient in a selected timeperiod following the hypoxic event may be averaged to determine thebaseline biomarker concentration for that patient. As will be understoodby those of ordinary skill in the art, in such cases, any sample whichhas a concentration level that significantly differs from the otherconcentration levels during that period of time (e.g., a datapoint/outlier having a concentration which deviates significantly fromthe other concentrations measured during the time frame) may be excludedfrom the averaging calculation. In some cases, if a sample has aconcentration which differs by greater than about 50% from thecalculated average, that sample may be excluded from the averagingcalculation. In some cases, if a sample has a concentration whichdiffers by more than about 0.5 pg/mL, about 1 pg/mL, about 2 pg/mL,about 5 pg/mL, or about 10 pg/mL from the calculated average, thatsample may be excluded from the averaging calculation. An anomalous datapoint may be observed and/or caused by administration of drugs to thepatient. The baseline biomarker concentration may be determined byaveraging the concentration of the biomarker in the samples obtainedfrom the patient between the first sample (e.g., obtained within 6 hoursof the hypoxic event) and 24 hours following the first collection of asample, or between the first sample and 18 hours following the firstcollection of a sample, or between the first sample, and 12 hoursfollowing the first collection of a sample, or between the first sample,and 6 hours following the first collection of a sample (e.g., notincluding any outliers).

The term “area under the curve,” (or AUC) is a common parameter used toanalyze data, and such calculations will be well known and understood bythose of ordinary skill in the art. In context with the methods of thisdisclosure, the AUC refers to the area under the biomarker concentrationversus time curve. Generally the calculation takes into account thebaseline biomarker concentration. Those of ordinary skill it the artwill be aware of suitable algorithms and/or computer programs capable ofdetermining the area under the curve for a selected set of data. In somecases, the area under the curve may be determined for more than oneportion of the data. For example, a first area under the curve value maybe determined for a first range of data, and a second area under thecurve value may be determined for a second range of data. The first andthe second ranges of data may or may not be overlapping (e.g., maycomprise some overlapping data points or may comprise different datapoints). It should be understood, that determining the area under thecurve can be accomplished using a variety of techniques which will beknown to those of ordinary skill in the art, including but not limitedto, plotting the concentration versus time on a graph and determiningthe area under the line/curve (e.g., optionally with use of a computerprogram) and/or determining a functional relationship between theconcentration and time and integrating the data without requiringphysically plotting or drawing of a graph. As used herein, the phrase“determining the area under the curve of a graph of a biomarkerconcentration versus time” covers all of the above techniques and othersapplicable for determining the area or equivalent, and is not limited tophysically or electronically plotting the concentration versus time on agraph and determining the area under the line/curve.

The term “change in biomarker concentration” refers to the change (e.g.,increase or decrease) in concentration of the biomarker over the timeperiod in which the samples are collected. An increase in biomarkerconcentration may be calculated by subtracting the baseline biomarkerconcentration (e.g., as described herein) from the maximum biomarkerconcentration. The “maximum biomarker concentration” is the maximummeasured concentration of the biomarker measured in a single samplecollected over the duration of the sample collection. For example, ifthe duration of the sample collection is 72 hours, the maximumconcentration is equal to the maximum measured concentration in a singlesample which was obtained from the patient in the 72 hour period. Adecrease in biomarker concentration may be calculated by subtracting anelevated biomarker concentration (e.g., as described herein) from theminimum biomarker concentration.

The term “magnitude of the fold increase of biomarker concentration”refers to the magnitude of the fold-increase in concentration over theduration of sample collection, and may be calculated by dividing themaximum biomarker concentration (e.g., as described herein) by thebaseline biomarker concentration (e.g., as described herein).

The term “duration of rise of biomarker concentration” refers to thetime over which the concentration of the biomarker is increasing duringthe time period over which the samples are collected. In some cases, theduration of rise of biomarker concentration can be determined bysubtracting the time at which the last sample was collected atapproximately the baseline biomarker concentration (e.g., starting timeof the rise) from the time at which the maximum biomarker concentrationwas observed (e.g., ending time of the rise). That is, the duration ofthe rise may be equal to the ending time of the rise minus the startingtime of the rise. The duration of rise is generally provided in hours.

The term “maximum slope of increasing biomarker concentration” refers tothe slope at which the maximum increase in concentration occurred overthe time period in which the samples were collected. Those of ordinaryskill will be aware of methods to calculate this value using commongraphical analysis methods. For example, a plot may be prepared showingconcentration versus time, and the maximum slope may be calculated basedon this plot. In some cases, the maximum slope is equal to the slopebetween two data points, whereas in other cases, the maximum slope maybe determined based on the average slope between a plurality of datapoints.

The above parameters, alone or in combination, may be correlated to aprognostic indication and/or a method of treatment for a patientfollowing a brain injury (e.g., caused by a hypoxic event). Followingdetermination of a correlation between biomarker concentrations and aprognostic indication and/or a method of treatment (e.g., using aplurality of samples obtained from a plurality of test patients withknown outcomes), the correlation can be used in connection with methodsto determine prognosis (e.g., prognostic indications) and/or methods oftreatment for patients with unknown outcomes. That is, an algorithm canbe developed relating changes in a biomarker concentration and specificmethods of treatment and/or prognostic indications using samples fromtest patients having been subject to known methods of treatment and/orhaving a known prognosis. Once the algorithm has been developed, it canbe used to determine methods of treatment and/or prognostic indicationsfor patients with unknown outcomes.

To determine a correlation between a biomarker and a prognosticindication and/or a method of treatment (e.g., to develop an algorithmrelating the measured biomarker concentration to preferred methods oftreatment and/or prognostic indications), a plurality of samples from aplurality of test patients may be obtained. A “test patient” is apatient who has a known outcome (e.g., a good or a poor CPC score)and/or has received a certain treatment. The plurality of the samplesobtained from each of the test patients may be analyzed to determine themeasure of the concentration of at least one biomarker in the each ofthe samples. Some or all of the parameters described herein may bedetermined for each patient, and the data may be analyzed to determinecorrelations between the parameters and the patient outcomes and/ortreatments, which can in turn be used to develop an algorithm. Thealgorithm may then be applied to the concentration of the at least onebiomarker in samples obtained from a patient having an unknown outcometo determine a method of treatment and/or a prognostic indication forthat patient. A specific example of such an analysis is described below,and further details are provided in Example 1.

Similar analysis and methods may be used to correlate suitable methodsof treatment based upon the measured concentration(s) of a biomarker ina plurality of samples obtained from a patient. In some cases, themethod of treatment may comprise administering at least one therapeuticagent to the patient. For example, the therapeutic agent may be aneuroprotective drug. Other non-limiting methods of treatment includeadministration of anti-oxidants, hypothermia, blood thinning, andadministration of steroids (e.g., to help reduce brain swelling) (e.g.,see T. S. Richmond, Cerebral Resuscitation After Global Brain Ischemia:Linking Research to Practice, American Association of Critical-CareNurses Journal, May 1997, Volume 8, Number 2).

A therapeutic agent is generally administered in an amount effective toprovide a medically desirable result. The effective amount will varywith the particular condition being treated, the age and physicalcondition of the subject being treated, the severity of the condition,the duration of the treatment, the nature of the concurrent therapy (ifany), the specific route of administration and the like factors withinthe knowledge and expertise of the health care practitioner. In somecases, a therapeutic agent may reduce brain injuries resulting from thehypoxic event. In some cases, the method of treatment may involve achange in treatment, such as an increase or decrease in the dose of atherapeutic agent, a switch from one therapeutic agent to anothertherapeutic agent, an addition of another therapeutic agent to theexisting therapeutic agent, or a combination thereof. A switch from onetherapeutic agent to another may involve a switch to a therapeutic agentwith a high risk profile but where the likelihood of expected benefit isincreased.

In one embodiment, a method of the present invention for determining atreatment protocol for and/or a prognostic indication of a patient'srecovery from a brain injury (e.g., resulting from a hypoxic event)comprises performing an assay on a plurality of samples to determine ameasure of the concentration of tau protein in each sample anddetermining a prognostic indication of the patient's recovery from thebrain injury and/or a method of treatment based at least in part on themeasure of the concentration of tau protein present in the samples. Thesample, in some embodiments, is a blood sample from the patient and/orplasma and/or serum derived from the blood sample. In some cases, theconcentration of the tau protein in the samples is less than about orabout 1000 pg/mL, less than about or about 900 pg/mL, less than about orabout 800 pg/mL, less than about or about 700 pg/mL, less than about orabout 600 pg/mL, less than about or about 500 pg/mL, less than about orabout 400 pg/mL, less than about or about 300 pg/mL, less than about orabout 200 pg/mL, less than about or about 100 pg/mL, less than about orabout 50 pg/mL, less than about or about 30 pg/mL, less than about orabout 20 pg/mL, less than about or about 10 pg/mL, less than about orabout 5 pg/mL, less than about or about 1 pg/mL, or less. In some cases,the assay has a limit of quantification of less than about or about 100pg/mL, less than about or about 50 pg/mL, less than about or about 40pg/mL, less than about or about 30 pg/mL, less than about or about 20pg/mL, less than about or about 10 pg/mL, less than about or about 5pg/mL, less than about or about 4 pg/mL, less than about or about 3pg/mL, less than about or about 2 pg/mL, less than about or about 1pg/mL, less than about or about 0.8 pg/mL, less than about or about 0.7pg/mL, less than about or about 0.6 pg/mL, less than about or about 0.5pg/mL, less than about or about 0.4 pg/mL, less than about or about 0.3pg/mL, less than about or about 0.2 pg/mL, less than about or about 0.1pg/mL, less than about or about 0.05 pg/mL, less than about or about0.04 pg/mL, less than about or about 0.02 pg/mL, less than about orabout 0.01 pg/mL, or less. In some cases, the assay has a limit ofdetection of less than about or about 100 pg/mL, less than about orabout 50 pg/mL, less than about or about 40 pg/mL, less than about orabout 30 pg/mL, less than about or about 20 pg/mL, less than about orabout 10 pg/mL, less than about or about 5 pg/mL, less than about orabout 4 pg/mL, less than about or about 3 pg/mL, less than about orabout 2 pg/mL, less than about or about 1 pg/mL, less than about orabout 0.8 pg/mL, less than about or about 0.7 pg/mL, less than about orabout 0.6 pg/mL, less than about or about 0.5 pg/mL, less than about orabout 0.4 pg/mL, less than about or about 0.3 pg/mL, less than about orabout 0.2 pg/mL, less than about or about 0.1 pg/mL, less than about orabout 0.05 pg/mL, less than about or about 0.04 pg/mL, less than aboutor about 0.02 pg/mL, less than about or about 0.01 pg/mL, or less.

In one embodiment, a correlation was determined between certainparameters (e.g., area under the curve of a plot of tau proteinconcentration versus time) and a good (e.g., CPC rating 1 or 2) or apoor (e.g., CPC rating of 3, 4, or 5) prognostic indication. Todetermine the correlation, an assay was carried out on each of thesamples obtained from a plurality of test patients having undergone abrain injury (e.g., resulting from a hypoxic event) (e.g., patientshaving a known outcome and/or having undergone a certain method oftreatment following a brain injury). The first sample was taken within 6hours of the brain injury (e.g., resulting from a hypoxic event), andadditional samples were generally obtained at about 1, 2, 6, 12, 24, 48,and 72 hours following the first sample (and optionally, at 96 and/or108 hours). Each of the test patients had a known prognostic outcomeaccording to the CPC scale. For each test patient, a plot of themeasured concentration of tau protein in pg/mL time in hours wasprepared. The data was analyzed and the area under the curve of the tauprotein concentration (in pg/mL) versus time (in hours) was determinedfor a variety of time ranges, and was determined to correlate with a“good” or “poor” prognosis for the patients. In some embodiments, theplot of the concentration of the tau protein versus time showed twopeaks, one occurring mostly in the first 24 hours following the hypoxicevent, and one occurring at some point following the first 24 hoursfollowing the hypoxic event. Thus, the area under the curve wasdetermined for each patient for three ranges of time: 1) for the entireduration of the data collection, 2) for the first 24 hours, and 3) forthe second peak in tau protein concentration (if present). Generally,the baseline used to determine the area under the curve for 1) and 2)was set to zero, whereas the baseline used to determine the area underthe curve for 3) was set as the concentration of tau protein determinedat the beginning of the rise of the second peak. For the total areaunder the curve, a value of greater than about 800 correlated with apoor prognosis (e.g., a CPC score of 3, 4, or 5) and a value of lessthan about 800 correlated with a good prognosis (e.g., a CPC score of 1or 2). For the area under the curve of the second peak, a value ofgreater than about 500 correlated with a poor prognosis (e.g., a CPCscore of 3, 4, or 5) and a value of less than about 500 correlated witha good prognosis (e.g., a CPC score of 1 or 2). Accordingly, acorrelation/algorithm was established between the varying parametersrelating to concentration and a prognostic indication.

The developed correlation/algorithm can be applied to patients withunknown outcomes. That is, samples may be obtained from a patient andthe concentration of tau protein in each of the samples can bedetermined. The data may be analyzed to determine the total area underthe curve and/or the area under the curve of the second tau proteinpeak. If the sum of the total area under the curve and/or the area underthe curve of the second peak is greater than 800 or 500, respectively,the prognostic indication for that patient is “poor,” and if the sum isless than 800 or 500, respectively, the prognostic indication is “good.”Using similar techniques, a variety of correlations may be determinedfor this tau protein (e.g., increase in tau protein concentration,duration of the increase of tau protein concentration, and/or themagnitude of the fold increase of tau protein) and/or other biomarkers.

Exemplary Assay Methods and Systems

Those of ordinary skill in the art will be aware of a variety of assaymethods and systems that may be used in connection with the methods ofthe present invention. Generally, the methods employed have low limitsof detection and/or limits of quantification as compared to bulkanalysis techniques (e.g., ELISA methods). The use of assay methods thathave low limits of detection and/or limits of quantification allows forcorrelations to be made between the various parameters discussed aboveand a method of treatment and/or diagnostic indication that mayotherwise not be determinable and/or apparent. For example, in themethod described above which correlates the total area under the curveand/or the area under the curve of a second peak of tau proteinconcentration to a prognostic indication of brain injury, the limits ofdetection, and/or limits of quantification needs to be substantiallylower than the LOD and/or LOQ provided by common ELISA techniques.

The terms “limit of detection” (or LOD) and “limit of quantification”(or LOQ) are given their ordinary meaning in the art. The LOD refers tothe lowest analyte concentration likely to be reliably distinguishedfrom background noise and at which detection is feasible. The LOD asused herein is defined as three standard deviations (SD) abovebackground noise. The LOQ refers to the lowest concentration at whichthe analyte can not only be reliably detected but at which somepredefined goals for bias and imprecision are met. Generally, as is usedherein, the LOQ refers to the lowest concentration above the LOD whereinthe coefficient of variation (CV) of the measured concentrations lessthan about 20%.

In some cases, an assay method employed has a limit of detection and/ora limit of quantification of less than about or about 500 pg/mL, lessthan about or about 250 pg/mL, less than about or about 100 pg/mL, lessthan about or about 50 pg/mL, less than about or about 40 pg/mL, lessthan about or about 30 pg/mL, less than about or about 20 pg/mL, lessthan about or about 10 pg/mL, less than about or about 5 pg/mL, lessthan about or about 4 pg/mL, less than about or about 3 pg/mL, less thanabout or about 2 pg/mL, less than about or about 1 pg/mL, less thanabout or about 0.8 pg/mL, less than about or about 0.7 pg/mL, less thanabout or about 0.6 pg/mL, less than about or about 0.5 pg/mL, less thanabout or about 0.4 pg/mL, less than about or about 0.3 pg/mL, less thanabout or about 0.2 pg/mL, less than about or about 0.1 pg/mL, less thanabout or about 0.05 pg/mL, less than about or about 0.04 pg/mL, lessthan about or about 0.02 pg/mL, less than about or about 0.01 pg/mL, orless. In some cases, an assay method employed has a limit ofquantification and/or a limit of detection between about 100 pg/mL andabout 0.01 pg/mL, between about 50 pg/mL and about 0.02 pg/mL, betweenabout 25 pg/mL and about 0.02 pg/mL, between about 10 pg/mL and about0.02 pg/mL. As will be understood by those of ordinary skill the art,the LOQ and/or LOD may differ for each assay method and/or eachbiomarker determined with the same assay. In some embodiments, the LODof an assay employed for detecting tau protein is about equal to or lessthan 0.02 pg/mL. In some embodiments, the LOQ for an assay employed fordetecting tau protein is equal to or less than 0.04 pg/mL.

In some embodiments, the concentration of biomarker molecules in thefluid sample that may be substantially accurately determined is lessthan about or about 5000 fM, less than about or about 3000 fM, less thanabout or about 2000 fM, less than about or about 1000 fM, less thanabout or about 500 fM, less than about or about 300 fM, less than aboutor about 200 fM, less than about or about 100 fM, less than about orabout 50 fM, less than about or about 25 fM, less than about or about 10fM, less than about or about 5 fM, less than about or about 2 fM, lessthan about or about 1 fM, less than about or about 0.5 fM, less thanabout or about 0.1 fM, or less. In some embodiments, the concentrationof biomarker molecules in the fluid sample that may be substantiallyaccurately determined is between about 5000 fM and about 0.1 fM, betweenabout 3000 fM and about 0.1 fM, between about 1000 fM and about 0.1 fM,between about 1000 fM and about 1 fM, between about 100 fM and about 1fM, between about 100 fM and about 0.1 fM, or the like. Theconcentration of analyte molecules or particles in a fluid sample may beconsidered to be substantially accurately determined if the measuredconcentration of the biomarker molecules in the fluid sample is withinabout 10% of the actual (e.g., true) concentration of the biomarkermolecules in the fluid sample. In certain embodiments, the measuredconcentration of the biomarker molecules in the fluid sample may bewithin about 5%, within about 4%, within about 3%, within about 2%,within about 1%, within about 0.5%, within about 0.4%, within about0.3%, within about 0.2% or within about 0.1%, of the actualconcentration of the biomarker molecules in the fluid sample. In somecases, the measure of the concentration determined differs from the true(e.g., actual) concentration by no greater than about 20%, no greaterthan about 15%, no greater than 10%, no greater than 5%, no greater than4%, no greater than 3%, no greater than 2%, no greater than 1%, or nogreater than 0.5%. The accuracy of the assay method may be determined,in some embodiments, by determining the concentration of biomarkermolecules in a fluid sample of a known concentration using the selectedassay method.

In some embodiments, an assay method employs a step of spatiallysegregating biomarker molecules into a plurality of locations tofacilitate detection/quantification, such that each locationcomprises/contains either zero or one or more biomarker molecules.Additionally, in some embodiments, the locations may be configured in amanner such that each location can be individually addressed. In someembodiments, a measure of the concentration of biomarker molecules in afluid sample may be determined by detecting biomarker moleculesimmobilized with respect to a binding surface having affinity for atleast one type of biomarker molecule. In certain embodiments the bindingsurface may form (e.g., a surface of a well/reaction vessel on asubstrate) or be contained within (e.g., a surface of a capture object,such as a bead, contained within a well) one of a plurality of locations(e.g., a plurality of wells/reaction vessels) on a substrate (e.g.,plate, dish, chip, optical fiber end, etc.). At least a portion of thelocations may be addressed and a measure indicative of thenumber/percentage/fraction of the locations containing at least onebiomarker molecule may be made. In some cases, based upon thenumber/percentage/fraction, a measure of the concentration of biomarkermolecules in the fluid sample may be determined. The measure of theconcentration of biomarker molecules in the fluid sample may bedetermined by a digital analysis method/system optionally employingPoisson distribution adjustment and/or based at least in part on ameasured intensity of a signal, as will be known to those of ordinaryskill in the art. In some cases, the assay methods and/or systems may beautomated.

Certain methods and systems which employ spatially segregating analytemolecules (e.g., biomarkers) are known in the art, and are described inU.S. Patent Application Publication No. US-2007-0259448 (Ser. No.11/707,385), filed Feb. 16, 2007, entitled “METHODS AND ARRAYS FORTARGET ANALYTE DETECTION AND DETERMINATION OF TARGET ANALYTECONCENTRATION IN SOLUTION,” by Walt et al.; U.S. Patent ApplicationPublication No. US-2007-0259385 (Ser. No. 11/707,383), filed Feb. 16,2007, entitled “METHODS AND ARRAYS FOR DETECTING CELLS AND CELLULARCOMPONENTS IN SMALL DEFINED VOLUMES,” by Walt et al.; U.S. PatentApplication Publication No. US-2007-0259381 (Ser. No. 11/707,384), filedFeb. 16, 2007, entitled “METHODS AND ARRAYS FOR TARGET ANALYTE DETECTIONAND DETERMINATION OF REACTION COMPONENTS THAT AFFECT A REACTION,” byWalt et al.; International Patent Publication No. WO 2009/029073(International Patent Application No. PCT/US2007/019184), filed Aug. 30,2007, entitled “METHODS OF DETERMINING THE CONCENTRATION OF AN ANALYTEIN SOLUTION,” by Walt et al.; U.S. Patent Application Publication No.US-2010-0075862 (Ser. No. 12/236,484), filed Sep. 23, 2008, entitled“HIGH SENSITIVITY DETERMINATION OF THE CONCENTRATION OF ANALYTEMOLECULES OR PARTICLES IN A FLUID SAMPLE,” by Duffy et al.; U.S. PatentApplication Publication No. US-2010-00754072 (Ser. No. 12/236,486),filed Sep. 23, 2008, entitled “ULTRA-SENSITIVE DETECTION OF MOLECULES ONSINGLE MOLECULE ARRAYS,” by Duffy et al.; U.S. Patent ApplicationPublication No. US-2010-0075439 (Ser. No. 12/236,488), filed Sep. 23,2008, entitled “ULTRA-SENSITIVE DETECTION OF MOLECULES BYCAPTURE-AND-RELEASE USING REDUCING AGENTS FOLLOWED BY QUANTIFICATION,”by Duffy et al.; International Patent Publication No. WO2010/039179(International Patent Application No. PCT/US2009/005248), filed Sep. 22,2009, entitled “ULTRA-SENSITIVE DETECTION OF MOLECULES OR ENZYMES,” byDuffy et al.; U.S. Patent Application Publication No. US-2010-0075355(Ser. No. 12/236,490), filed Sep. 23, 2008, entitled “ULTRA-SENSITIVEDETECTION OF ENZYMES BY CAPTURE-AND-RELEASE FOLLOWED BY QUANTIFICATION,”by Duffy et al.; U.S. patent application Ser. No. 12/731,130, filed Mar.24, 2010, published as US-2011-0212848 on Sep. 1, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OROTHER CAPTURE OBJECTS,” by Duffy et al.; International PatentApplication No. PCT/US2011/026645, filed Mar. 1, 2011, published as WO2011/109364 on Sep. 9, 2011, entitled “ULTRA-SENSITIVE DETECTION OFMOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” by Duffyet al.; International Patent Application No. PCT/US2011/026657, filedMar. 1, 2011, published as WO 2011/109372 on Sep. 9, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS,”by Duffy et al.; U.S. patent application Ser. No. 12/731,135, filed Mar.24, 2010, published as US-2011-0212462 on Sep. 1, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS,”by Duffy et al.; International Patent Application No. PCT/US2011/026665,filed Mar. 1, 2011, published as WO 2011/109379 on Sep. 9, 2011,entitled “METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FORTHE DETECTION OF MOLECULES OR PARTICLES,” by Rissin et al.; U.S. patentapplication Ser. No. 12/731,136, filed Mar. 24, 2010, published asUS-2011-0212537 on Sep. 1, 2011, entitled “METHODS AND SYSTEMS FOREXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OF MOLECULES ORPARTICLES,” by Duffy et al.; U.S. patent application Ser. No.13/035,472, filed Feb. 25, 2011, entitled “SYSTEMS, DEVICES, AND METHODSFOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES,” by Fournier etal.; U.S. patent application Ser. No. 13/037,987, filed Mar. 1, 2011,published as US-2011-0245097 on Oct. 6, 2011, entitled “METHODS ANDSYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OFMOLECULES OR PARTICLES,” by Rissin et al.; each herein incorporated byreference.

Additional details of exemplary, non-limiting assay methods whichcomprise one or more steps of spatially segregating biomarker moleculeswill now be described. In certain embodiments, a method for detectionand/or quantifying biomarker molecules in a sample comprisesimmobilizing a plurality of biomarker molecules with respect to aplurality of capture objects (e.g., beads) that each include a bindingsurface having affinity for at least one type of biomarker. For example,the capture objects may comprise a plurality of beads comprising aplurality of capture components (e.g., an antibody having specificaffinity for a biomarker of interest, etc.). At least some of thecapture objects (e.g., at least some associated with at least onebiomarker molecule) may be spatially separated/segregated into aplurality of locations, and at least some of the locations may beaddressed/interrogated (e.g., using an imaging system). A measure of theconcentration of biomarker molecules in the fluid sample may bedetermined based on the information received when addressing thelocations (e.g., using the information received from the imaging systemand/or processed using a computer implemented control system). In somecases, a measure of the concentration may be based at least in part onthe number of locations determined to contain a capture object that isor was associated with at least one biomarker molecule. In other casesand/or under differing conditions, a measure of the concentration may bebased at least in part on an intensity level of at least one signalindicative of the presence of a plurality of biomarker molecules and/orcapture objects associated with a biomarker molecule at one or more ofthe addressed locations.

In some embodiments, the number/percentage/fraction of locationscontaining a capture object but not containing a biomarker molecule mayalso be determined and/or the number/percentage/fraction of locationsnot containing any capture object may also be determined. In suchembodiments, a measure of the concentration of biomarker molecules inthe fluid sample may be based at least in part on the ratio of thenumber of locations determined to contain a capture object associatedwith a biomarker molecule to the total number of locations determined tocontain a capture object not associated with a biomarker molecule,and/or a measure of the concentration of biomarker molecule in the fluidsample may be based at least in part on the ratio of the number oflocations determined to contain a capture object associated with abiomarker molecule to the number of locations determined to not containany capture objects, and/or a measure of the concentration of biomarkermolecule in the fluid sample may be based at least in part on the ratioof the number of locations determined to contain a capture objectassociated with a biomarker molecule to the number of locationsdetermined to contain a capture object. In yet other embodiments, ameasure of the concentration of biomarker molecules in a fluid samplemay be based at least in part on the ratio of the number of locationsdetermined to contain a capture object and a biomarker molecule to thetotal number of locations addressed and/or analyzed.

In certain embodiments, at least some of the plurality of captureobjects (e.g., at least some associated with at least one biomarkermolecule) are spatially separated into a plurality of locations, forexample, a plurality of reaction vessels in an array format. Theplurality of reaction vessels may be formed in, on and/or of anysuitable material, and in some cases, the reaction vessels can be sealedor may be formed upon the mating of a substrate with a sealingcomponent, as discussed in more detail below. In certain embodiments,especially where quantization of the capture objects associated with atleast one biomarker molecule is desired, the partitioning of the captureobjects can be performed such that at least some (e.g., a statisticallysignificant fraction; e.g., as described in International PatentApplication No. PCT/US2011/026645, filed Mar. 1, 2011, published as WO2011/109364 on Sep. 9, 2011, entitled “ULTRA-SENSITIVE DETECTION OFMOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” by Duffyet al) of the reaction vessels comprise at least one or, in certaincases, only one capture object associated with at least one biomarkermolecule and at least some (e.g., a statistically significant fraction)of the reaction vessels comprise an capture object not associated withany biomarker molecules. The capture objects associated with at leastone biomarker molecule may be quantified in certain embodiments, therebyallowing for the detection and/or quantification of biomarker moleculesin the fluid sample by techniques described in more detail herein.

An exemplary assay method may proceed as follows. A sample fluidcontaining or suspected of containing biomarker molecules is provided.An assay consumable comprising a plurality of assay sites is exposed tothe sample fluid. In some cases, the biomarker molecules are provided ina manner (e.g., at a concentration) such that a statisticallysignificant fraction of the assay sites contain a single biomarkermolecule and a statistically significant fraction of the assay sites donot contain any biomarker molecules. The assay sites may optionally beexposed to a variety of reagents (e.g., using a reagent loader) and orrinsed. The assay sites may then optionally be sealed and imaged (see,for example, U.S. patent application Ser. No. 13/035,472, filed Feb. 25,2011, entitled “SYSTEMS, DEVICES, AND METHODS FOR ULTRA-SENSITIVEDETECTION OF MOLECULES OR PARTICLES,” by Fournier et al.). The imagesare then analyzed (e.g., using a computer implemented control system)such that a measure of the concentration of the biomarker molecules inthe fluid sample may be obtained, based at least in part, bydetermination of the number/fraction/percentage of assay sites whichcontain a biomarker molecule and/or the number/fraction/percentage ofsites which do not contain any biomarkers molecules. In some cases, thebiomarker molecules are provided in a manner (e.g., at a concentration)such that at least some assay sites comprise more than one biomarkermolecule. In such embodiments, a measure of the concentration ofbiomarker molecules in the fluid sample may be obtained at least in parton an intensity level of at least one signal indicative of the presenceof a plurality of biomarkers molecules at one or more of the assay sites

In some cases, the methods optionally comprise exposing the fluid sampleto a plurality of capture objects, for example, beads. At least some ofthe biomarker molecules are immobilized with respect to a bead. In somecases, the biomarker molecules are provided in a manner (e.g., at aconcentration) such that a statistically significant fraction of thebeads associate with a single biomarker molecule and a statisticallysignificant fraction of the beads do not associate with any biomarkermolecules. At least some of the plurality of beads (e.g., thoseassociated with a single biomarker molecule or not associated with anybiomarker molecules) may then be spatially separated/segregated into aplurality of assay sites (e.g., of an assay consumable). The assay sitesmay optionally be exposed to a variety of reagents and/or rinsed. Atleast some of the assay sites may then be addressed to determine thenumber of assay sites containing a biomarker molecule. In some cases,the number of assay sites containing a bead not associated with abiomarker molecule, the number of assay sites not containing a beadand/or the total number of assay sites addressed may also be determined.Such determination(s) may then be used to determine a measure of theconcentration of biomarker molecules in the fluid sample. In some cases,more than one biomarker molecule may associate with a bead and/or morethan one bead may be present in an assay site. In some cases, theplurality biomarker molecules may be exposed to at least one additionalreaction component prior to, concurrent with, and/or following spatiallyseparating at least some of the biomarker molecules into a plurality oflocations.

The biomarker molecules may be directly detected or indirectly detected.In the case of direct detection, a biomarker molecule may comprise amolecule or moiety that may be directly interrogated and/or detected(e.g., a fluorescent entity). In the case of indirect detection, anadditional component is used for determining the presence of thebiomarker molecule. For example, the biomarker molecules (e.g.,optionally associated with a bead) may be exposed to at least one typeof binding ligand. A “binding ligand,” is any molecule, particle, or thelike which specifically binds to or otherwise specifically associateswith a biomarker molecule to aid in the detection of the biomarkermolecule. In certain embodiments, a binding ligand may be adapted to bedirectly detected (e.g., the binding ligand comprises a detectablemolecule or moiety) or may be adapted to be indirectly detected (e.g.,including a component that can convert a precursor labeling agent into alabeling agent). A component of a binding ligand may be adapted to bedirectly detected in embodiments where the component comprises ameasurable property (e.g., a fluorescence emission, a color, etc.). Acomponent of a binding ligand may facilitate indirect detection, forexample, by converting a precursor labeling agent into a labeling agent(e.g., an agent that is detected in an assay). A “precursor labelingagent” is any molecule, particle, or the like, that can be converted toa labeling agent upon exposure to a suitable converting agent (e.g., anenzymatic component). A “labeling agent” is any molecule, particle, orthe like, that facilitates detection, by acting as the detected entity,using a chosen detection technique. In some embodiments, the bindingligand may comprise an enzymatic component (e.g., horseradishperoxidase, beta-galactosidase, alkaline phosphatase, etc.). A firsttype of binding ligand may or may not be used in conjunction withadditional binding ligands (e.g., second type, etc.).

More than one type of binding may be employed in any given assay method,for example, a first type of binding ligand and a second type of bindingligand. In one example, the first type of binding ligand is able toassociate with a first type of biomarker molecule and the second type ofbinding ligand is able to associate with the first binding ligand. Inanother example, both a first type of binding ligand and a second typeof binding ligand may associate with the same or different epitopes of asingle biomarker molecule, as described herein. In some embodiments, atleast one binding ligand comprises an enzymatic component.

In some embodiments, a binding ligand and/or a biomarker may comprise anenzymatic component. The enzymatic component may convert a precursorlabeling agent (e.g., an enzymatic substrate) into a labeling agent(e.g., a detectable product). A measure of the concentration ofbiomarker molecules in the fluid sample can then be determined based atleast in part by determining the number of locations containing alabeling agent (e.g., by relating the number of locations containing alabeling agent to the number of locations containing a biomarkermolecule (or number of capture objects associated with at least onebiomarker molecule to total number of capture objects)). Non-limitingexamples of enzymes or enzymatic components include horseradishperoxidase, beta-galactosidase, and alkaline phosphatase. Othernon-limiting examples of systems or methods for detection includeembodiments where nucleic acid precursors are replicated into multiplecopies or converted to a nucleic acid that can be detected readily, suchas the polymerase chain reaction (PCR), rolling circle amplification(RCA), ligation, Loop-Mediated Isothermal Amplification (LAMP), etc.Such systems and methods will be known to those of ordinary skill in theart, for example, as described in “DNA Amplification: CurrentTechnologies and Applications,” Vadim Demidov et al., 2004.

Another exemplary embodiment of indirect detection is as follows. Insome cases, the biomarker molecules may be exposed to a precursorlabeling agent (e.g., enzymatic substrate) and the enzymatic substratemay be converted to a detectable product (e.g., fluorescent molecule)upon exposure to a biomarker molecule.

The assay methods and systems may employ a variety of differentcomponents, steps, and/or other aspects that will be known andunderstood by those of ordinary skill in the art. For example, a methodmay further comprise determining at least one background signaldetermination (e.g., and further comprising subtracting the backgroundsignal from other determinations), wash steps, and the like. In somecases, the assays or systems may include the use of at least one bindingligand, as described herein. In some cases, the measure of theconcentration of biomarker molecules in a fluid sample is based at leastin part on comparison of a measured parameter to a calibration curve. Insome instances, the calibration curve is formed at least in part bydetermination at least one calibration factor, as described above.

As will be understood by those of ordinary skill in the art, a systemand/or method may be calibrated using natural and/or synthetic forms ofthe target biomarker, and/or analogues thereof. In embodiments where thetarget analyte is a tau protein, the system and/or method may becalibrated using one or more natural and/or synthetic isoforms of tauprotein. Naturally occurring tau proteins are described herein.Synthetic isoforms of tau proteins include two nearest neighbor antibodyepitope synthetic peptides (<20 amino acids) to long synthetic peptides(80-100 amino acids in length. In some cases, short isoforms of thenaturally occurring tau proteins may be employed. For example, tauprotein isoforms can have varying lengths of amino acids selected fromthe tau 441 sequence. Non-limiting examples of short forms of tauproteins include tau 50-mers (e.g., comprising residues 187-237,190-240, or 155-205 of tau 441) and tau 64-mers (e.g., comprisingresidues 155-235 of tau 441, RGAAP PGQKG QTPPA PKpTPP SSKSG DRSGY SSPGSPGTSR TPSLP TPPTR EPKKV AVVRT PPKS-NH2 (SEQ ID NO.: 1)). In some cases,at least a portion of the tau protein(s) used may be phosphorylated(e.g., RGAAP PGQKG QTPPA PKpTPP SSKSG DRSGY SSPGS PGTSR TPSLP TPPTREPKKV AVVRpT PPKS-NH2 (SEQ ID NO.: 2)).

In certain embodiments, solubilized, or suspended precursor labelingagents may be employed, wherein the precursor labeling agents areconverted to labeling agents which are insoluble in the liquid and/orwhich become immobilized within/near the location (e.g., within thereaction vessel in which the labeling agent is formed). Such precursorlabeling agents and labeling agents and their use is described incommonly owned U.S. Patent Application Publication No. US-2010-0075862(Ser. No. 12/236,484), filed Sep. 23, 2008, entitled “HIGH SENSITIVITYDETERMINATION OF THE CONCENTRATION OF ANALYTE MOLECULES OR PARTICLES INA FLUID SAMPLE,” by Duffy et al., incorporated herein by reference.

An exemplary embodiment of an assay method that may be used in certainembodiments of the invention is illustrated in FIG. 1a . A plurality ofcapture objects 2, are provided (step (A)). In this particular example,the plurality of capture objects comprises a plurality of beads. Thebeads are exposed to a fluid sample containing a plurality of biomarkermolecules 3 (e.g., beads 2 are incubated with biomarker molecules 3). Atleast some of the biomarker molecules are immobilized with respect to abead. In this example, the biomarker molecules are provided in a manner(e.g., at a concentration) such that a statistically significantfraction of the beads associate with a single biomarker molecule and astatistically significant fraction of the beads do not associate withany biomarker molecules. For example, as shown in step (B), biomarkermolecule 4 is immobilized with respect to bead 5, thereby formingcomplex 6, whereas some beads 7 are not associated with any biomarkermolecules. It should be understood, in some embodiments, more than onebiomarker molecule may associate with at least some of the beads, asdescribed herein. At least some of the plurality of beads (e.g., thoseassociated with a single biomarker molecule or not associated with anybiomarker molecules) may then be spatially separated/segregated into aplurality of locations. As shown in step (C), the plurality of locationsis illustrated as substrate 8 comprising a plurality of wells/reactionvessels 9. In this example, each reaction vessel comprises either zeroor one beads. At least some of the reaction vessels may then beaddressed (e.g., optically or via other detection means) to determinethe number of locations containing a biomarker molecule. For example, asshown in step (D), the plurality of reaction vessels are interrogatedoptically using light source 15, wherein each reaction vessel is exposedto electromagnetic radiation (represented by arrows 10) from lightsource 15. The light emitted (represented by arrows 11) from eachreaction vessel is determined (and/or recorded) by detector 15 (in thisexample, housed in the same system as light source 15). The number ofreaction vessels containing a biomarker molecule (e.g., reaction vessels12) is determined based on the light detected from the reaction vessels.In some cases, the number of reaction vessels containing a bead notassociated with a biomarker molecule (e.g., reaction vessel 13), thenumber of wells not containing a bead (e.g., reaction vessel 14) and/orthe total number of wells addressed may also be determined. Suchdetermination(s) may then be used to determine a measure of theconcentration of biomarker molecules in the fluid sample.

A non-limiting example of an embodiment where a capture object isassociated with more than one biomarker molecule is illustrated in FIG.1b . A plurality of capture objects 20 are provided (step (A)). In thisexample, the plurality of capture objects comprises a plurality ofbeads. The plurality of beads is exposed to a fluid sample containingplurality of biomarker molecules 21 (e.g., beads 20 are incubated withbiomarker molecules 21). At least some of the biomarker molecules areimmobilized with respect to a bead. For example, as shown in step (B),biomarker molecule 22 is immobilized with respect to bead 24, therebyforming complex 26. Also illustrated is complex 30 comprising a beadimmobilized with respect to three biomarker molecules and complex 32comprising a bead immobilized with respect to two biomarker molecules.Additionally, in some cases, some of the beads may not associate withany biomarker molecules (e.g., bead 28). The plurality of beads fromstep (B) is exposed to a plurality of binding ligands 31. As shown instep (C), a binding ligand associates with some of the biomarkermolecules immobilized with respect to a bead. For example, complex 40comprises bead 34, biomarker molecule 36, and binding ligand 38. Thebinding ligands are provided in a manner such that a statisticallysignificant fraction of the beads comprising at least one biomarkermolecule become associated with at least one binding ligand (e.g., one,two, three, etc.) and a statistically significant fraction of the beadscomprising at least one biomarker molecule do not become associated withany binding ligands. At least a portion of the plurality of beads fromstep (C) are then spatially separated into a plurality of locations. Asshown in step (D), in this example, the locations comprise a pluralityof reaction vessels 41 on a substrate 42. The plurality of reactionvessels may be exposed to the plurality of beads from step (C) such ateach reaction vessel contains zero or one beads. The substrate may thenbe analyzed to determine the number of reaction vessels containing abinding ligand (e.g., reaction vessels 43), wherein in the number may berelated to a measure of the concentration of biomarker molecules in thefluid sample. In some cases, the number of reaction vessels containing abead and not containing a binding ligand (e.g., reaction vessel 44), thenumber of reaction vessels not containing a bead (e.g., reaction vessel45), and/or the total number of reaction vessels addressed/analyzed mayalso be determined. Such determination(s) may then be used to determinea measure of the concentration of biomarker molecules in the fluidsample.

In some embodiments, a plurality of locations may be addressed and/or aplurality of capture objects and/or species/molecules/particles ofinterest may be detected substantially simultaneously. “Substantiallysimultaneously” when used in this context, refers toaddressing/detection of the locations/captureobjects/species/molecules/particles of interest at approximately thesame time such that the time periods during which at least twolocations/capture objects/species/molecules/particles of interest areaddressed/detected overlap, as opposed to being sequentiallyaddressed/detected, where they would not. Simultaneousaddressing/detection can be accomplished by using various techniques,including optical techniques (e.g., CCD detector). Spatially segregatingcapture objects/species/molecules/particles into a plurality ofdiscrete, resolvable locations, according to some embodimentsfacilitates substantially simultaneous detection by allowing multiplelocations to be addressed substantially simultaneously. For example, forembodiments where individual species/molecules/particles are associatedwith capture objects that are spatially segregated with respect to theother capture objects into a plurality of discrete, separatelyresolvable locations during detection, substantially simultaneouslyaddressing the plurality of discrete, separately resolvable locationspermits individual capture objects, and thus individualspecies/molecules/particles (e.g., biomarker molecules) to be resolved.For example, in certain embodiments, individual molecules/particles of aplurality of molecules/particles are partitioned across a plurality ofreaction vessels such that each reaction vessel contains zero or onlyone species/molecule/particle. In some cases, at least about 80%, atleast about 85%, at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, atleast about 99.5% of all species/molecules/particles are spatiallyseparated with respect to other species/molecules/particles duringdetection. A plurality of species/molecules/particles may be detectedsubstantially simultaneously within a time period of less than about 1second, less than about 500 milliseconds, less than about 100milliseconds, less than about 50 milliseconds, less than about 10milliseconds, less than about 1 millisecond, less than about 500microseconds, less than about 100 microseconds, less than about 50microseconds, less than about 10 microseconds, less than about 1microsecond, less than about 0.5 microseconds, less than about 0.1microseconds, or less than about 0.01 microseconds, less than about0.001 microseconds, or less. In some embodiments, the plurality ofspecies/molecules/particles may be detected substantially simultaneouslywithin a time period of between about 100 microseconds and about 0.001microseconds, between about 10 microseconds and about 0.01 microseconds,or less.

In some embodiments, the locations are optically interrogated. Thelocations exhibiting changes in their optical signature may beidentified by a conventional optical train and optical detection system.Depending on the detected species (e.g., type of fluorescence entity,etc.) and the operative wavelengths, optical filters designed for aparticular wavelength may be employed for optical interrogation of thelocations. In embodiments where optical interrogation is used, thesystem may comprise more than one light source and/or a plurality offilters to adjust the wavelength and/or intensity of the light source.In some embodiments, the optical signal from a plurality of locations iscaptured using a CCD camera.

In some embodiments of the present invention, the plurality of reactionvessels may be sealed (e.g., after the introduction of the biomarkermolecules, binding ligands, and/or precursor labeling agent), forexample, through the mating of the second substrate and a sealingcomponent. The sealing of the reaction vessels may be such that thecontents of each reaction vessel cannot escape the reaction vesselduring the remainder of the assay. In some cases, the reaction vesselsmay be sealed after the addition of the biomarker molecules and,optionally, at least one type of precursor labeling agent to facilitatedetection of the biomarker molecules. For embodiments employingprecursor labeling agents, by sealing the contents in some or eachreaction vessel, a reaction to produce the detectable labeling agentscan proceed within the sealed reaction vessels, thereby producing adetectable amount of labeling agents that is retained in the reactionvessel for detection purposes.

The plurality of locations may be formed may be formed using a varietyof methods and/or materials. In some embodiments, the plurality oflocations comprises a plurality of reaction vessels/wells on asubstrate. In some cases, the plurality of reaction vessels is formed asan array of depressions on a first surface. In other cases, however, theplurality of reaction vessels may be formed by mating a sealingcomponent comprising a plurality of depressions with a substrate thatmay either have a featureless surface or include depressions alignedwith those on the sealing component. Any of the device components, forexample, the substrate or sealing component, may be fabricated from acompliant material, e.g., an elastomeric polymer material, to aid insealing. The surfaces may be or made to be hydrophobic or containhydrophobic regions to minimize leakage of aqueous samples from themicrowells. The reactions vessels, in certain embodiments, may beconfigured to receive and contain only a single capture object.

In some embodiments, the reaction vessels may all have approximately thesame volume. In other embodiments, the reaction vessels may havediffering volumes. The volume of each individual reaction vessel may beselected to be appropriate to facilitate any particular assay protocol.For example, in one set of embodiments where it is desirable to limitthe number of capture objects used for biomarker capture contained ineach vessel to a small number, the volume of the reaction vessels mayrange from attoliters or smaller to nanoliters or larger depending uponthe nature of the capture objects, the detection technique and equipmentemployed, the number and density of the wells on the substrate and theexpected concentration of capture objects in the fluid applied to thesubstrate containing the wells. In one embodiment, the size of thereaction vessel may be selected such only a single capture object usedfor biomarker capture can be fully contained within the reaction vessel(see, for example, U.S. patent application Ser. No. 12/731,130, filedMar. 24, 2010, published as US-2011-0212848 on Sep. 1, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OROTHER CAPTURE OBJECTS,” by Duffy et al.; International PatentApplication No. PCT/US2011/026645, filed Mar. 1, 2011, published as WO2011/109364 on Sep. 9, 2011, entitled “ULTRA-SENSITIVE DETECTION OFMOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” by Duffyet al., each herein incorporated by reference).

In some embodiments, the reaction vessels may have a volume betweenabout 1 femtoliter and about 1 picoliter, between about 1 femtolitersand about 100 femtoliters, between about 10 attoliters and about 100picoliters, between about 1 picoliter and about 100 picoliters, betweenabout 1 femtoliter and about 1 picoliter, or between about 30femtoliters and about 60 femtoliters. In some cases, the reactionvessels have a volume of less than about 1 picoliter, less than about500 femtoliters, less than about 100 femtoliters, less than about 50femtoliters, or less than about 1 femtoliter. In some cases, thereaction vessels have a volume of about 10 femtoliters, about 20femtoliters, about 30 femtoliters, about 40 femtoliters, about 50femtoliters, about 60 femtoliters, about 70 femtoliters, about 80femtoliters, about 90 femtoliters, or about 100 femtoliters.

The total number of locations and/or density of the locations employedin an assay (e.g., the number/density of reaction vessels in an array)can depend on the composition and end use of the array. For example, thenumber of reaction vessels employed may depend on the number of types ofbiomarker molecule and/or binding ligand employed, the suspectedconcentration range of the assay, the method of detection, the size ofthe capture objects, the type of detection entity (e.g., free labelingagent in solution, precipitating labeling agent, etc.). Arrayscontaining from about 2 to many billions of reaction vessels (or totalnumber of reaction vessels) can be made by utilizing a variety oftechniques and materials. Increasing the number of reaction vessels inthe array can be used to increase the dynamic range of an assay or toallow multiple samples or multiple types of biomarker molecules to beassayed in parallel. The array may comprise between one thousand and onemillion reaction vessels per sample to be analyzed. In some cases, thearray comprises greater than one million reaction vessels. In someembodiments, the array comprises between about 1,000 and about 50,000,between about 1,000 and about 1,000,000, between about 1,000 and about10,000, between about 10,000 and about 100,000, between about 100,000and about 1,000,000, between about 100,000 and about 500,000, betweenabout 1,000 and about 100,000, between about 50,000 and about 100,000,between about 20,000 and about 80,000, between about 30,000 and about70,000, between about 40,000 and about 60,000 reaction vessels. In someembodiments, the array comprises about 10,000, about 20,000, about50,000, about 100,000, about 150,000, about 200,000, about 300,000,about 500,000, about 1,000,000, or more, reaction vessels.

The array of reaction vessels may be arranged on a substantially planarsurface or in a non-planar three-dimensional arrangement. The reactionvessels may be arrayed in a regular pattern or may be randomlydistributed. In a specific embodiment, the array is a regular pattern ofsites on a substantially planar surface permitting the sites to beaddressed in the X-Y coordinate plane.

In some embodiments, the reaction vessels are formed in a solidmaterial. As will be appreciated by those in the art, the number ofpotentially suitable materials in which the reaction vessels can beformed is very large, and includes, but is not limited to, glass(including modified and/or functionalized glass), plastics (includingacrylics, polystyrene and copolymers of styrene and other materials,polypropylene, polyethylene, polybutylene, polyurethanes, cyclic olefincopolymer (COC), cyclic olefin polymer (COP), Teflon®, polysaccharides,nylon or nitrocellulose, etc.), elastomers (such as poly(dimethylsiloxane) and poly urethanes), composite materials, ceramics, silica orsilica-based materials (including silicon and modified silicon), carbon,metals, optical fiber bundles, or the like. In general, the substratematerial may be selected to allow for optical detection withoutappreciable autofluorescence. In certain embodiments, the reactionvessels may be formed in a flexible material.

A reaction vessel in a surface (e.g., substrate or sealing component)may be formed using a variety of techniques known in the art, including,but not limited to, photolithography, stamping techniques, moldingtechniques, etching techniques, or the like. As will be appreciated bythose of the ordinary skill in the art, the technique used can depend onthe composition and shape of the supporting material and the size andnumber of reaction vessels.

In a particular embodiment, an array of reaction vessels is formed bycreating microwells on one end of a fiber optic bundle and utilizing aplanar compliant surface as a sealing component. In certain suchembodiments, an array of reaction vessels in the end of a fiber opticbundle may be formed as follows. First, an array of microwells is etchedinto the end of a polished fiber optic bundle. Techniques and materialsfor forming and etching a fiber optic bundle are known to those ofordinary skill in the art. For example, the diameter of the opticalfibers, the presence, size and composition of core and cladding regionsof the fiber, and the depth and specificity of the etch may be varied bythe etching technique chosen so that microwells of the desired volumemay be formed. In certain embodiments, the etching process createsmicrowells by preferentially etching the core material of the individualglass fibers in the bundle such that each well is approximately alignedwith a single fiber and isolated from adjacent wells by the claddingmaterial. Potential advantages of the fiber optic array format is thatit can produce thousands to millions of reaction vessels withoutcomplicated microfabrication procedures and that it can provide theability to observe and optically address many reaction vesselssimultaneously.

Each microwell may be aligned with an optical fiber in the bundle sothat the fiber optic bundle can carry both excitation and emission lightto and from the wells, enabling remote interrogation of the wellcontents. Further, an array of optical fibers may provide the capabilityfor simultaneous or non-simultaneous excitation of molecules in adjacentvessels, without signal “cross-talk” between fibers. That is, excitationlight transmitted in one fiber does not escape to a neighboring fiber.

Alternatively, the equivalent structures of a plurality of reactionvessels may be fabricated using other methods and materials that do notutilize the ends of an optical fiber bundle as a substrate. For example,the array may be a spotted, printed or photolithographically fabricatedsubstrate produced by techniques known in the art; see for exampleWO95/25116; WO95/35505; PCT US98/09163; U.S. Pat. Nos. 5,700,637,5,807,522, 5,445,934, 6,406,845, and 6,482,593. In some cases, the arraymay be produced using molding, embossing, and/or etching techniques aswill be known to those of ordinary skill in the art.

In some embodiments, the plurality of locations may not comprise aplurality of reaction vessels/wells. For example, in embodiments wherecapture objects are employed, a patterned substantially planar surfacemay be employed and the patterned areas form a plurality of locations.In some cases, the patterned areas may comprise substantiallyhydrophilic surfaces which are substantially surrounded by substantiallyhydrophobic surfaces. In certain embodiments, a plurality of captureobjects (e.g., beads) may be substantially surrounded by a substantiallyhydrophilic medium (e.g., comprising water), and the beads may beexposed to the patterned surface such that the beads associate in thepatterned areas (e.g., the hydrophilic locations on the surface),thereby spatially segregating the plurality of beads. For example, inone such embodiment, a substrate may be or include a gel or othermaterial able to provide a sufficient barrier to mass transport (e.g.,convective and/or diffusional barrier) to prevent capture objects usedfor biomarker capture and/or precursor labeling agent and/or labelingagent from moving from one location on or in the material to anotherlocation so as to cause interference or cross-talk between spatiallocations containing different capture objects during the time framerequired to address the locations and complete the assay. For example,in one embodiment, a plurality of capture objects is spatially separatedby dispersing the capture objects on and/or in a hydrogel material. Insome cases, a precursor labeling agent may be already present in thehydrogel, thereby facilitating development of a local concentration ofthe labeling agent (e.g., upon exposure to a binding ligand or biomarkermolecule carrying an enzymatic component). As still yet anotherembodiment, the capture objects may be confined in one or morecapillaries. In some cases, the plurality of capture objects may beabsorbed or localized on a porous or fibrous substrate, for example,filter paper. In some embodiments, the capture objects may be spatiallysegregated on a uniform surface (e.g., a planar surface), and thecapture objects may be detected using precursor labeling agents whichare converted to substantially insoluble or precipitating labelingagents that remain localized at or near the location of where thecorresponding capture object is localized. The use of such substantiallyinsoluble or precipitating labeling agents is described herein. In somecases, single biomarker molecules may be spatially segregated into aplurality of droplets. That is, single biomarker molecules may besubstantially contained in a droplet containing a first fluid. Thedroplet may be substantially surrounded by a second fluid, wherein thesecond fluid is substantially immiscible with the first fluid.

In some embodiments, during the assay, at least one washing step may becarried out. In certain embodiments, the wash solution is selected sothat it does not cause appreciable change to the configuration of thecapture objects and/or biomarker molecules and/or does not disrupt anyspecific binding interaction between at least two components of theassay (e.g., a capture component and a biomarker molecule). In othercases, the wash solution may be a solution that is selected tochemically interact with one or more assay components. As will beunderstood by those of ordinary skill in the art, a wash step may beperformed at any appropriate time point during the inventive methods.For example, a plurality of capture objects may be washed after exposingthe capture objects to one or more solutions comprising biomarkermolecules, binding ligands, precursor labeling agents, or the like. Asanother example, following immobilization of the biomarker moleculeswith respect to a plurality of capture objects, the plurality of captureobjects may be subjected to a washing step thereby removing anybiomarker molecules not specifically immobilized with respect to acapture object.

Other assay methods in addition to those described herein are known inthe art and may be used in connection with the inventive methods. Forexample, various analyzers are commercially available for thedetermination of the concentration of biomarkers. The assay methodsemployed should meet the algorithm requirements for LOD and LOQ.

The following examples are included to demonstrate various features ofthe invention. Those of ordinary skill in the art should, in light ofthe present disclosure, will appreciate that many changes can be made inthe specific embodiments which are disclosed while still obtaining alike or similar result without departing from the scope of the inventionas defined by the appended claims. Accordingly, the following examplesare intended only to illustrate certain features of the presentinvention, but do not necessarily exemplify the full scope of theinvention.

Example 1

The following example provides experimental details relating toprognostication of neurological outcome in comatose survivors of oxygendeprivation, using tau proteins

Objective: Use of peripheral tau protein measurements as an indicator ofthe presence of brain injury has remained elusive, in large part due tothe lack of adequate sensitivity for reliable measurement of the proteinin serum or plasma using common technologies. Additionally, little wasknown about the time-dependence of tau protein release from the centralnervous system into peripheral circulation. Using methods describedherein, which, in certain embodiments are capable of ultra sensitive tauprotein measurement, serial serum samples from 25 resuscitated cardiacarrest patients were analyzed to longitudinally study tau proteinrelease into serum and determine prognostic significance of tau proteinelevation for prediction of long-term cognitive impairment from hypoxicinsult.

Summary of Methods: 25 unconscious patients with cardiac arrest wereresuscitated with restoration of spontaneous circulation and admitted toa hospital intensive care unit (ICU). Patients were treated withhypothermia and repeated blood samplings were obtained during the firstfive days in the ICU. Serum levels of total tau protein were measuredwith a digital immunoassay described in Example 2. Cognitive assessmentswere made using Cerebral Performance Categorization (CPC) at dischargefrom the ICU and six months later. Tau protein data were analyzed in thecontext of six-month cognitive outcome.

Summary of Results: Tau protein elevations ranged from modest to veryhigh, and exhibited unexpected bi-modal kinetic profiles in manypatients. Total area-under-the-curve (AUC) was highly prognostic forsix-month cognitive outcome. AUC of the secondary tau protein peakexhibited 100% sensitivity and 91% specificity for predicting 6-monthoutcome.

Summary of Conclusions: The data indicate that sufficiently sensitiveperipheral tau protein measurements in conjunction with an understandingof tau protein release kinetics have clinical utility for brain injuryassessment and prognostication of cognitive outcome.

Additional Background: Tau proteins, with a molecular mass of 48 to 67kd depending on isoform, are associated with microtubules and localizedin the axonal compartment of neurons. Tau proteins plays a structuralrole in the assembly of tubulin monomers into axonal microtubulebundles, which are important for maintaining the cytoskeleton and axonaltransport. Tau protein is generally elevated in the cerebrospinal fluid(CSF) of patients with neurodegenerative disease and severe headinjuries, making it a candidate for peripheral measurement as abiomarker of acquired or traumatic brain injury (ABI, TBI). However,studies on peripheral tau protein have been hampered by its lowabundance in serum and plasma (typically low pg/mL), making itsmeasurement difficult.

While CSF tau protein elevation is known to correlate with 1-yearoutcome in severe TBI patients, no such correlation has been made toserum tau protein, in part because most common assays cannot accuratelydetect the low levels of tau protein in serum. In addition, the clinicalvalue of serum tau protein for assessment of minor head injury has beenquestioned. A previous study looked at serum tau protein elevationmeasured in 24 ischemic stroke patients studied using an immunoassaywith a limit of detection of 60 pg/mL, but no correlation was made orfound between tau protein appearance to stroke severity. A recent ratTBI model indicated serum tau protein elevation peaked rapidly anddeclined after six hours, with no significant additional tau proteinelevation over 7 days. Little else has been reported about the kineticsof tau protein movement across the blood brain barrier (BBB), nor thepotential prognostic significance of peripheral tau protein appearancewith ABI or TBI.

Methods: This example employed the protocol described in Example 2below, which is capable in certain embodiments of three logs greatersensitivity than typical conventional methods. The assay was utilized toexamine serial serum samples from 25 resuscitated cardiac arrestpatients to longitudinally study tau protein release into serum andprobe for prognostic significance of tau protein elevation forprediction of long-term cognitive impairment due to hypoxic insult.

The study was performed at the general intensive care unit at UppsalaUniversity Hospital, Sweden, and approved by the Human Ethics Committeeof Uppsala, Sweden. Twenty-five unconscious patients with cardiac arrestwere resuscitated with restoration of spontaneous circulation (ROSC).Patients were >18 yrs old, exhibited systolic blood pressure >80 mmHgafter ROSC, and a Glasgow Coma Scale≤7.

Upon admission, hypothermia treatment was started immediately afterresuscitation. Ventilation was administered during the coma period, witha target PaO₂ of ≥12 kPa (90 mmHg) and PaCO₂ between 5.0 and 5.5 kPa(38-41 mmHg). Targeted mean arterial pressure was 65-100 mmHg, withapplication of inotropic/vasopressor support, if required, usingdobutamine as the first line medication, followed by noradrenaline(norepinephrine) or adrenaline (epinephrine), if necessary. If thepatient was considered euvolaemic but had a diuresis of less than 0.5ml/kg/h, furosemide was administered. Furosemide was also given if theintensive care physician considered that the patient had a fluidoverload. All patients received an arterial line in the radial orfemoral artery for blood sampling. Serial blood samples were collected,starting as soon as possible in the emergency phase (within 6 h aftercardiac arrest), and continuing at 1, 2, 6, 12, 24, 48, 72, 96, and 108h after cardiac arrest. Serum aliquots were frozen at −70° C. untilanalysis.

Patient outcome was assessed in accordance with the Glasgow-Pittsburghcerebral performance category (CPC) scale at discharge from theintensive care unit and 6 months later. The CPC scale ranges from 1 to5, with 1 representing mildest possible neurological deficit (patient isable to return to work), and 4-5 representing the most severe deficit(vegetative) and death. A CPC of 1 or 2 was considered a “good” outcomeand a CPC score of 3-5 a “poor” outcome. For patients who died after ICUdischarge, the better of the two scores was used, as recommended by theUtstein templates.

Patient serum samples were measured in triplicate by a single moleculedigital immunoassay for tau protein (see Example 2 for more details).The technique involves performing a paramagnetic bead-based ELISA usingbeta-galactosidase as a reporter, followed by isolation of individualcapture beads within femtoliter-sized reaction wells in a microarray.Isolation of individual beads permits the buildup of fluorescentsubstrate in the presence of tau protein, such that wells containing asingle immunocomplex can be detected. The limit of detection of theassay is 0.02 pg/mL, making it approximately 1000-fold more sensitivethan typical conventional immunoassays. The assay was calibrated from 0pg/mL to 100 pg/mL total tau protein and was able to precisely measureserum tau protein in the patient samples. The extreme sensitivity of themethod permits pre-dilution of the samples prior to assay, reducingpotential endogenous interferences. All samples were pre-diluted 1:4with a PBS-tween diluent prior to assay.

Tau protein elevation profiles were analyzed for area-under-the-curve(AUC) with GraphPad Prism 5.0d (GraphPad Software, La Jolla, Calif.).Four of the 25 patients died 24-48 hours after admission, and thesepatients were excluded from AUC analysis. AUC was evaluated during thefirst 24-hour period as well as over the full time course of samples (to108 hours) using a baseline of zero. In addition, the AUC of secondarytau protein elevation peaks were estimated assuming a baselinecorresponding to the tau protein concentration measured at the initialtime point of the secondary peak. Statistical significance between‘good’ and ‘poor’ 6-month outcome was determined by student t-test, withsignificance taken as p<0.05.

Results: Representative elevation profiles for patients with good andpoor 6-month outcomes are depicted in FIG. 2. FIG. 2 shows serum taufollowing resuscitation from cardiac arrest. CPC scores are listing foreach patient in the legends. The first two numbers correspond to the CPCassessments upon discharge from the ICU and six months later. The thirdnumber represents overall 6-month outcome assessment (1=poor, 0=good).FIGS. 2a-2c depict representative groupings of patients exhibitingdifferent profiles of tau elevations: a) initial 24-hour peaks; b)delayed peaks, and c) both initial 24-hour and delayed peaks. FIG. 2ddepicts representative profiles from patients with good outcomes. Notethe difference in scales. Error bars depict SD of triplicatemeasurements.

Tau protein expression ranged from almost undetectable to largeelevations approaching 700 pg/mL. There was a strong general associationbetween tau protein elevation and patient outcome: the more tau proteinexpressed, the greater the likelihood for poor 6-month outcome. Tauprotein elevation also showed clear bi-modal tendencies, with theappearance of one or both peaks varying with patient. The initial peakwas generally fully expressed during the first 24 hours followingcardiac arrest, while the secondary peak generally expressed after 24-48hours. Some patients exhibited only the first peak (FIG. 2a ), someexhibited only the second peak (FIG. 2b ), and some exhibited both peaks(FIG. 2c ).

TABLE 2 AUC </= 24 hr AUC all AUC 2^(nd) peak only Patient Good PoorGood Poor Good Poor 2 1590 4759 1440 3 708.9 2145 0 4 748.3 2861 154.2 5625.2 3007 1890 6 179.9 1232 897.5 7 4.72 92.86 54.5 8 11.19 1646 1545 959 1141 0 10 109.7 9336 8206 11 14.63 84.56 0 12 52.15 848.2 567.8 1326.01 288.5 215.8 14 12.93 431.5 393 15 12.31 184.4 168.2 17 0.02 30.6830.9 18 15.78 46.62 0 20 334.6 6827 5511 21 580.8 19875 18051 23 103926150 23935 24 12.92 72.9 35.94 25 9.37 8987 8842 AVG 160.26 413.17730.61 7521.83 101.66 6447.39 t-test (p) 0.090609467 0.0117269460.01197362

To compare the significance of the primary and secondary elevationprofiles for 6-month outcome, AUCs were calculated for the first 24hours (referred to as the “first peak”), the full time course, and thesecondary peak only. Table 2 exhibits AUCs for each patient, which areplotted in FIG. 3. More specifically, Table 2 tabulates characteristicsof serum tau elevation profiles from resuscitated survivors of cardiacarrest during the first 96-108 hours following admission to the ICD.Parameters were sorted on the basis of good or poor 6-month cerebraloutcome and compared by student t-test. Four patients (all with goodoutcome) exhibited no discernable secondary peaks. FIG. 3 plots AUCresults a) across the first 24 hours, b) for all serial samplings, andc) the secondary tau peak only. Error bars depict standard error of themeans.

Weak correlation was observed between the initial 24 hours and 6-monthoutcome (p>0.05). Notably, there was a strong correlation between thesecondary peak and 6-month outcome (p=0.01). The weak correlationbetween initial tau protein appearance and outcome reflectsinter-patient differences in primary peak expression rather than lack ofsignificance between tau protein elevation and outcome. Calculation ofoverall AUC gave similar statistical significance with outcome as theAUC of the second peak only. Patients with good outcome generally hadvery low serum tau protein, and secondary peaks where either absent orweak (FIG. 2d ).

The appearance and magnitude of the secondary tau protein peak washighly prognostic for 6-month outcome. Bifurcating the data with an AUCcut point of 500 resulted in 100% sensitivity (10/10 patients) and 91%specificity (10/11 patients) for predicting good and poor 6-monthoutcomes respectively.

Discussion: These data represent a high-resolution longitudinalexamination of serum tau protein elevation following an acute ABI event.The bimodal profile elevation kinetics are consistent with two modes ofneuronal damage: initially upon acute oxygen deprivation, followed bydelayed cell death due to apoptosis and/or cerebral swelling. With ROSCas an inclusion criterion, the elevation kinetics should be unrelated toreperfusion differences between patients. Patients were all treated withhypothermia, and were not treated with drugs known to significantlyaffect BBB permeability. It seems likely the bimodal profiles arerelated to neuronal damage rather than BBB or reperfusion variables.

Inter-patient differences in expression one or both elevation peakscould be related to the extent and duration of the hypoxia. Globalcerebral ischemia could trigger rapid necrosis in addition tolonger-term apoptosis cascades. Sub lethal hypoxic encephalopathy canset a series of toxic reactions in motion that finish off injuredneurons and kill additional ones over hours or days following theinsult. While early tau protein peaks were less prognostics for 6-monthoutcome than the secondary elevation, they are nonetheless deadly whenhigh levels of tau protein are measured. Among the four patients who didnot survive the first 48 hours, two patients had prominent initial tauprotein peaks of well over 200 pg/mL that had dropped 10-fold by 24hours (not shown). In these patients, it may have been that the acuteinitial necrosis was sufficiently lethal, and it might be anticipatedthat survival would have witnessed prominent secondary peaks.

It is noted that the serial sampling in this study was concluded at 108hours. It may be that additional tau protein elevation occurs beyond 108hours. Studies of tau protein elevation in CSF following severe TBI haverevealed temporal elevations well beyond five days. It is possible thatpatients in the present study exhibiting tau protein peaks in the first24 hours with minimal secondary elevation could go on to expresssignificant additional tau protein beyond 108 hours.

Since the magnitude of cognitive impairment should reflect the magnitudeof hypoxia, correlation of serum tau protein elevation with cognitiveoutcome indicates released tau protein reflects the extent of hypoxiaand associated neuronal damage. Serum tau protein appearance as measuredby digital immunoassay exhibited considerable prognostic significancefor 6-month cognitive outcome, with a sensitivity and specificity of100% using an AUC cut point of 500. This example demonstrates thatserial blood measurements of tau protein in the ICU followingresuscitation from cardiac arrest has a clinical value for stratifyinglikely cognitive outcome.

Example 2

The following example describes the tau protein ultra-sensitive digitalimmunoassay for plasma tau using single molecule arrays that wasemployed in Example 1.

Reagents were developed for a paramagnetic bead-based ELISA. Tau proteinmolecules in plasma were captured on antibody-coated paramagneticcapture beads and labeled with an enzyme conjugate. The beads wereloaded into arrays of 50,000, 50-femtoliter reaction wells etched intobundles of optical fibers. Single capture beads trapped in each wellwere sealed in the presence of enzyme substrate and imaged using afluorescence microscope fitted with a CCD camera. At low concentrations,the images were analyzed for the presence or absence of singleimmunocomplexes of labeled tau protein, resulting in a digital signal.At high concentrations, the analog intensity of the beads was normalizedto the digital signals, extending the dynamic range of the assay to overfour logs. Analytical performance of the assay was evaluated, and serumsamples from hypoxia patients were tested for tau protein.

The assay described in this example has a detection limit (i.e., LOD) of0.02 pg/mL and was linear to 100 pg/mL tau protein (R2>0.996, Patient J.J cal curve). Results using serum samples from hypoxia patients showed abi-phasic response across the time course post hypoxic insult.

Concentrations of tau protein in serum and plasma are believed to beover 100-fold lower than in cerebrospinal fluid.

This example describes the development and validation of a digitalimmunoassay using single molecule array technology that is capable ofmeasuring tau protein in hypoxia induced serum without biomarkerenrichment or sample pretreatment procedures. The assay exhibits over1000-fold greater sensitivity than validated commercially availableELISAs. The assay can be used for directly measuring and monitoringserum tau protein in therapeutic trials aimed at altering and loweringlevels of this protein, down to sub-femtomolar levels.

The single molecule array technology employed two primary steps: aninitial analyte capture step conducted with paramagnetic beads, followedby isolation of individual beads in arrays of femtoliter-sized reactionwells for digital imaging. Isolation of the individual beads inmicrowells permits the buildup of fluorescent product from the enzymelabel such that signal from a single immunocomplex is readily detectedusing a CCD camera. This approach permits counting of single moleculeswhen tau protein concentrations are low enough that the ratio of boundlabeled peptide per bead is much less than one. In this concentrationrealm, Poisson statistics predict that bead-containing microwells in thearray will contain either a single labeled tau protein molecule or nolabeled tau protein molecules, resulting in a binary signal. Due to theamplified sensitivity for detecting label molecules afforded byconfining fluorescent product buildup to the microwells, concentrationsof label (detector anti-Tau antibody and enzyme label) can be reducedrelative to standard ELISAs. Lowered concentrations of labeling reagentsreduces their interaction with capture beads, resulting in reducednonspecific binding enabling high signal to background ratios, even atextremely low concentrations of biomarker. For higher biomarkerconcentrations where all beads contain one or more labeledimmunocomplexes, digital signals from the Poisson realm are used tocalibrate analog intensity measurements, extending the dynamic range toover four logs.

Three reagents were developed for this tau protein immunoassay: capturebeads, biotinylated detector, and a conjugate ofstreptavidin:beta-galactosidase The capture bead reagent comprised of acommercially available monoclonal anti-tau antibody (Covance) directedto an epitope (amino acids 210-230). The antibody was covalentlyattached by standard coupling chemistry to 2.7 μm carboxy paramagneticmicrobeads (Varian). Because individual beads were captured in arraywells 4.5 μm wide×3.25 μm deep, it was advantageous that the capturebeads remain monomeric. The antibody-coated beads were diluted to aworking concentration of 6×106 beads/mL in Tris buffer with a surfactantand BSA. The biotinylated detector reagent was comprised of twocommercially available monoclonal anti-tau antibodys (Pierce) directedto the N-terminus of the Capture Detector (amino acids 159-163 and194-198). The antibodies were biotinylated using standard methods(Solulink), and the biotinylation level was confirmedspectrophotometrically per manufacturer's instructions. The monomericstate of the detector antibody before and after biotinylation wasconfirmed by size exclusion HPLC. The biotinylated detector antibodieswere diluted to individual concentration of 0.2 μg/ml for assay in a PBSdiluent containing a surfactant and newborn calf serum, NCS (PBS/NCS).The enzyme conjugate—streptavidin: β-galactosidase (SβG)—was prepared bycovalent conjugation of purified streptavidin (Thermo Scientific) andβ-galactosidase (Sigma) using standard coupling chemistry(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, ThermoScientific). Aliquots of a concentrated stock solution of SβG wereprepared in PBS with 50% glycerol and transferred to −20° C. forstorage. Prior to assay, an aliquot was thawed and diluted to 25 pM inPBS/NCS with MgCl2. Purified Tau 381 antigen for calibrators and forspecificity testing were from Millipore.

Assay calibrators and controls were prepared by dilution of Tau 381stock in PBS diluent containing a surfactant and BSA (PBS/BSA). In somecases, a stock solution prepared by dilution to 1000 ng/mL in PBS/10G.For dynamic range/linearity characterization, a series of calibratorswere prepared by serial 3-fold dilution to give a calibration range of0-100 pg/mL. To evaluate assay day-to-day reproducibility at lowconcentrations, three low controls were prepared at 0.1, 5.0, and 50.0pg/mL in PBS/BSA.

50,000 well optical fiber microarrays were prepared as previouslydescribed. In brief, optical fiber bundles (Schott North America) werecut into 5 cm lengths and sequentially polished with 30, 9, and 1um-sized diamond lapping films. One end of each bundle was etched in a0.025 N HCl solution for 2 minutes and then submerged into water. Thedifferential etch rate of the core and cladding glass of the fiberbundles caused an array of 4.5 um diameter wells to be formed in thecore fibers.

Bead-plasma incubations and labeling of immunocomplexes in conical 96well plates (Axygen) were conducted using a robotic liquid handlingsystem (Tecan EVO 150). Conical wells are used to facilitate magneticattraction of the beads to the sides of the wells for efficient removalof reaction mixtures and bead washing. For magnetic attraction, amicroplate bar magnet (Invitrogen) was used. Incubation periods wereconducted with shaking on a microplate shaker (VWR) to keep beadssuspended in the wells. The assays were initiated by mixing 100 uL ofsample with 600,000 capture beads, and the mixtures were incubated withshaking for two hours. Serum samples were pre-diluted 1:4 prior to assaywith PBS/BSA as a precaution for sample quality and interferenceeffects. Following serum incubation in the presence of biotinylateddetector antibody, the beads were washed 3 times with a wash buffer of5-fold concentrated PBS with a surfactant (5×PBS). After a the washstep, 100 μL of streptavidin-β-galactosidase was incubated with thebeads for 30 minutes to form the final enzyme-labeled immunocomplex. Thebeads were then washed eight times per above, and concentrated to 2×107beads/ml with the addition of a reduced volume (30 μL) of array loadingbuffer comprised of PBS with a surfactant. Beads were then loaded ontothe arrays. 10 μL of the concentrated bead solution (2×106 beads) werepipetted onto the arrays and the arrays were centrifuged at 1,300 g for10 minutes. Excess beads were removed by a PBS rinse and swabbing withdeionized water. With this technique, array filling by the beads wasgenerally 50-60%, which was adequate for minimizing contributions toimprecision from Poisson noise. Wells containing tau protein-labeledbeads were detected utilizing beta-galactosidase catalyzed hydrolysis ofresorufin β-D-galactopyranoside (RGP, Invitrogen) into fluorescentproduct (resorufin, excitation 558 nm, emission 577 nm). To introduceRGP substrate to the array wells, droplets of substrate were placed on asilicone gasket and introduced into the array wells with a mechanicalplatform. This step resulted in an array of sealed femtoliter wells inwhich enzyme-containing beads developed a concentrated fluorescencesignal.

Imaging was accomplished via a custom-built fluorescence imaging systemcontaining a light source, objectives, filter cubes and a CCD camera.For each sample, five fluorescent images of one second each wereacquired (to identify wells containing an enzyme) and one white lightimage was acquired (to identify wells containing a bead). Backgroundfluorescence and any contaminating artifacts were discriminated fromtrue ‘positive’ wells by analyzing for signal growth over the multipleimages. These images were analyzed to determine the average number ofenzymes per bead (AEB) across the concentration range. In some cases, at<50% active beads, the system was determined to be in the digital realm,so AEB was be determined from the fraction of beads that contain atleast one enzyme and the Poisson distribution; and at >50% active beads,the average fluorescence intensity of the beads was normalized to theaverage fluorescence intensity of beads containing a single enzyme toyield AEB. In other cases, at <70% active beads relative to total beads,AEB was determined as a count of active beads corrected for a lowstatistical probability of multiple enzymes per bead; and at >70% activebeads, the probability of multiple enzymes/bead increases such that allwells contain multiple enzymes and all are growing in signal and in thisrealm, the signal is was no longer digital, and average fluorescenceintensities of the wells were converted to AEB based on the averageintensities of wells containing single enzymes as determined at lowerAβ42 concentrations. The AEB unit worked continuously across the digitaland analog realms.

For description of various details associate with this assay, see, forexample, U.S. patent application Ser. No. 12/731,130, filed Mar. 24,2010, published as US-2011-0212848 on Sep. 1, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES USING BEADS OROTHER CAPTURE OBJECTS,” by Duffy et al.; International PatentApplication No. PCT/US2011/026645, filed Mar. 1, 2011, published as WO2011/109364 on Sep. 9, 2011, entitled “ULTRA-SENSITIVE DETECTION OFMOLECULES OR PARTICLES USING BEADS OR OTHER CAPTURE OBJECTS,” by Duffyet al.; International Patent Application No. PCT/US2011/026657, filedMar. 1, 2011, published as WO 2011/109372 on Sep. 9, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS,”by Duffy et al.; U.S. patent application Ser. No. 12/731,135, filed Mar.24, 2010, published as US-2011-0212462 on Sep. 1, 2011, entitled“ULTRA-SENSITIVE DETECTION OF MOLECULES USING DUAL DETECTION METHODS,”by Duffy et al.; International Patent Application No. PCT/US2011/026665,filed Mar. 1, 2011, published as WO 2011/109379 on Sep. 9, 2011,entitled “METHODS AND SYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FORTHE DETECTION OF MOLECULES OR PARTICLES,” by Rissin et al.; U.S. patentapplication Ser. No. 12/731,136, filed Mar. 24, 2010, published asUS-2011-0212537 on Sep. 1, 2011, entitled “METHODS AND SYSTEMS FOREXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OF MOLECULES ORPARTICLES,” by Duffy et al.; U.S. patent application Ser. No.13/035,472, filed Feb. 25, 2011, entitled “SYSTEMS, DEVICES, AND METHODSFOR ULTRA-SENSITIVE DETECTION OF MOLECULES OR PARTICLES,” by Fournier etal.; U.S. patent application Ser. No. 13/037,987, filed Mar. 1, 2011,published as US-2011-0245097 on Oct. 6, 2011, entitled “METHODS ANDSYSTEMS FOR EXTENDING DYNAMIC RANGE IN ASSAYS FOR THE DETECTION OFMOLECULES OR PARTICLES,” by Rissin et al.; each herein incorporated byreference. While several embodiments of the present invention have beendescribed and illustrated herein, those of ordinary skill in the artwill readily envision a variety of other means and/or structures forperforming the functions and/or obtaining the results and/or one or moreof the advantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

What is claimed:
 1. A method of producing a bodily fluid sample of apatient suspected of having a neurological condition containing ananalytically quantified amount of endogenous tau protein, said methodcomprising: (A) obtaining a volume of bodily fluid comprising blood, ora blood component selected from plasma and serum, (B) diluting thevolume of bodily fluid, and (C) quantifying through the use of ananalytical protein concentration measurement assay a concentration oftau protein in the volume of bodily fluid to produce the bodily fluidsample containing the analytically quantified amount of endogenous tauprotein; wherein the analytically quantified concentration of endogenoustau protein contained in the bodily fluid sample is less than about 5pg/nil, and a limit of quantification of the analytical proteinconcentration measurement assay used in step (C) for quantifying theconcentration of tau protein is less than about 0.2 pg/mL.
 2. The methodof claim 1, wherein the analytical protein concentration measurementassay used in step (C) comprises: i. exposing a plurality of captureobjects that each include a binding surface having affinity for tauprotein molecules to the volume of bodily fluid; ii. immobilizing atleast a portion of the tau protein molecules with respect to theplurality of capture objects such that at least some of the captureobjects associate with at least one tau protein molecule and astatistically significant fraction of the capture objects do notassociate with any tau protein molecules; iii. determining a proportionof the plurality of capture objects that associated with a tau proteinmolecule in step ii; and iv. determining a measure of tau proteinmolecule concentration in the volume of bodily fluid based at least inpart on the proportion determined in step iii.
 3. The method of claim 2,wherein the plurality of capture objects comprises a plurality of beads.4. The method of claim 1, wherein the analytical protein concentrationmeasurement assay used in step (C) comprises spatially segregating tauprotein molecules into a plurality of locations, wherein a statisticallysignificant fraction of the locations contain a single tau proteinmolecule and a statistically significant fraction of the locations donot contain any tau protein molecules.
 5. The method of claim 4, whereinthe plurality of locations comprises a plurality of reaction vessels. 6.The method of claim 1, wherein each of the tau protein moleculesquantified in step (C) is selected from the group consisting of tau 23(352, 0N3R), tau 2.4 (383, 0N4R), tau 37 (381, 1N3R), tau 34 (412,1N4R), tau 39 (410, 2N3R), and tau 40 (441, 2N4R).
 7. The method ofclaim 6, wherein at least a portion of the tau protein molecules arephosphorylated.
 8. The method of claim 1, wherein the volume of bodilyfluid comprises blood.
 9. The method of claim 1, wherein the volume ofbodily fluid comprises plasma.
 10. The method of claim 1, wherein thevolume of bodily fluid comprises serum.
 11. The method of claim 1,wherein the volume of bodily fluid does not comprise cerebrospinal fluid(CSF).
 12. The method of claim 1, wherein the analytical proteinconcentration measurement assay used in step (C) comprises: i.suspending a plurality of capture objects that each include a bindingsurface having affinity for tau protein molecules in the diluted volumeof bodily fluid, ii. immobilizing at least a portion of the tau proteinmolecules with respect to at least some of the plurality of captureobjects suspended in the diluted volume of bodily fluid such that atleast some of the capture objects associate with at least one tauprotein molecule from the volume of bodily fluid, iii. interrogating atleast some of the capture objects associated with at least one taumolecule from the volume of bodily of fluid; and iv. determining ameasure of tau protein molecule concentration in the volume of bodilyfluid based at least in part on the interrogating step performed in stepiii.
 13. The method of claim 12, wherein the plurality of captureobjects comprises a plurality of beads.
 14. The method claim 1, whereinthe limit of detection for the analytical protein concentrationmeasurement assay is less than about 0.02 pg/mL.
 15. The method of claim1, wherein the patient suspected of having a neurological condition isrecovering from a brain injury.
 16. The method of claim 15, wherein thevolume of bodily fluid has been collected from the patient within 12hours of the brain injury.
 17. The method of claim 15, wherein the braininjury results from a hypoxic event.
 18. The method of claim 15 whereinthe brain injury comprises a hypoxic event caused by cardiac arrest. 19.The method of claim 15, wherein the brain injury comprises a hypoxicevent caused by stroke.
 20. The method of claim 15, wherein the braininjury comprises a hypoxic event caused by an ischemic event.
 21. Themethod of claim 15 wherein the brain injury comprises a hypoxic eventcaused by a thrombosis.